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Sample records for binding affinity prediction

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

    Science.gov (United States)

    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

  2. Blind prediction of host-guest binding affinities: A new SAMPL3 challenge

    OpenAIRE

    Muddana, Hari S.; Varnado, C. Daniel; Bielawski, Christopher W.; Urbach, Adam R.; Isaacs, Lyle; Geballe, Matthew T; Gilson, Michael K.

    2012-01-01

    The computational prediction of protein-ligand binding affinities is of central interest in early-stage drug-discovery, and there is a widely recognized need for improved methods. Low molecular weight receptors and their ligands—i.e. host-guest systems – represent valuable test-beds for such affinity prediction methods, because their small size makes for fast calculations and relatively facile numerical convergence. The SAMPL3 community exercise included the first ever blind prediction challe...

  3. Prediction of SAMPL3 Host-Guest Affinities with the Binding Energy Distribution Analysis Method (BEDAM)

    OpenAIRE

    Gallicchio, Emilio; Ronald M Levy

    2012-01-01

    BEDAM calculations are described to predict the free energies of binding of a series of anaesthetic drugs to a recently characterized acyclic cucurbituril host. The modeling predictions, conducted as part of the SAMPL3 host-guest affinity blind challenge, are generally in good quantitative agreement with the experimental measurements. The correlation coefficient between computed and measured binding free energies is 70% with high statistical significance. Multiple conformational stereoisomers...

  4. Predicting the Impact of Missense Mutations on Protein-Protein Binding Affinity.

    Science.gov (United States)

    Li, Minghui; Petukh, Marharyta; Alexov, Emil; Panchenko, Anna R

    2014-04-01

    The crucial prerequisite for proper biological function is the protein's ability to establish highly selective interactions with macromolecular partners. A missense mutation that alters the protein binding affinity may cause significant perturbations or complete abolishment of the function, potentially leading to diseases. The availability of computational methods to evaluate the impact of mutations on protein-protein binding is critical for a wide range of biomedical applications. Here, we report an efficient computational approach for predicting the effect of single and multiple missense mutations on protein-protein binding affinity. It is based on a well-tested simulation protocol for structure minimization, modified MM-PBSA and statistical scoring energy functions with parameters optimized on experimental sets of several thousands of mutations. Our simulation protocol yields very good agreement between predicted and experimental values with Pearson correlation coefficients of 0.69 and 0.63 and root-mean-square errors of 1.20 and 1.90 kcal mol(-1) for single and multiple mutations, respectively. Compared with other available methods, our approach achieves high speed and prediction accuracy and can be applied to large datasets generated by modern genomics initiatives. In addition, we report a crucial role of water model and the polar solvation energy in estimating the changes in binding affinity. Our analysis also reveals that prediction accuracy and effect of mutations on binding strongly depends on the type of mutation and its location in a protein complex. PMID:24803870

  5. A knowledge-guided strategy for improving the accuracy of scoring functions in binding affinity prediction

    Directory of Open Access Journals (Sweden)

    Wang Renxiao

    2010-04-01

    Full Text Available Abstract Background Current scoring functions are not very successful in protein-ligand binding affinity prediction albeit their popularity in structure-based drug designs. Here, we propose a general knowledge-guided scoring (KGS strategy to tackle this problem. Our KGS strategy computes the binding constant of a given protein-ligand complex based on the known binding constant of an appropriate reference complex. A good training set that includes a sufficient number of protein-ligand complexes with known binding data needs to be supplied for finding the reference complex. The reference complex is required to share a similar pattern of key protein-ligand interactions to that of the complex of interest. Thus, some uncertain factors in protein-ligand binding may cancel out, resulting in a more accurate prediction of absolute binding constants. Results In our study, an automatic algorithm was developed for summarizing key protein-ligand interactions as a pharmacophore model and identifying the reference complex with a maximal similarity to the query complex. Our KGS strategy was evaluated in combination with two scoring functions (X-Score and PLP on three test sets, containing 112 HIV protease complexes, 44 carbonic anhydrase complexes, and 73 trypsin complexes, respectively. Our results obtained on crystal structures as well as computer-generated docking poses indicated that application of the KGS strategy produced more accurate predictions especially when X-Score or PLP alone did not perform well. Conclusions Compared to other targeted scoring functions, our KGS strategy does not require any re-parameterization or modification on current scoring methods, and its application is not tied to certain systems. The effectiveness of our KGS strategy is in theory proportional to the ever-increasing knowledge of experimental protein-ligand binding data. Our KGS strategy may serve as a more practical remedy for current scoring functions to improve their

  6. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Lund, Ole

    2007-01-01

    correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the...

  7. Binding affinity prediction of novel estrogen receptor ligands using receptor-based 3-D QSAR methods.

    Science.gov (United States)

    Sippl, Wolfgang

    2002-12-01

    We have recently reported the development of a 3-D QSAR model for estrogen receptor ligands showing a significant correlation between calculated molecular interaction fields and experimentally measured binding affinity. The ligand alignment obtained from docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection procedure, a significant and robust model was obtained (q(2)(LOO)=0.921, SDEP=0.345). To further analyze the robustness and the predictivity of the established model several recently developed estrogen receptor ligands were selected as external test set. An excellent agreement between predicted and experimental binding data was obtained indicated by an external SDEP of 0.531. Two other traditionally used prediction techniques were applied in order to check the performance of the receptor-based 3-D QSAR procedure. The interaction energies calculated on the basis of receptor-ligand complexes were correlated with experimentally observed affinities. Also ligand-based 3-D QSAR models were generated using program FlexS. The interaction energy-based model, as well as the ligand-based 3-D QSAR models yielded models with lower predictivity. The comparison with the interaction energy-based model and with the ligand-based 3-D QSAR models, respectively, indicates that the combination of receptor-based and 3-D QSAR methods is able to improve the quality of prediction. PMID:12413831

  8. Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA

    OpenAIRE

    Veprintsev, Dmitry B.; Fersht, Alan R.

    2008-01-01

    The tumour suppressor p53 is a transcription factor that binds DNA in the vicinity of the genes it controls. The affinity of p53 for specific binding sites relative to other DNA sequences is an inherent driving force for specificity, all other things being equal. We measured the binding affinities of systematically mutated consensus p53 DNA-binding sequences using automated fluorescence anisotropy titrations. Based on measurements of the effects of every possible single base-pair substitution...

  9. Prediction of SAMPL3 Host-Guest Binding Affinities: Evaluating the Accuracy of Generalized Force-Fields

    OpenAIRE

    Muddana, Hari S.; Gilson, Michael K.

    2012-01-01

    We used the second-generation mining minima method (M2) to compute the binding affinities of the novel host-guest complexes in the SAMPL3 blind prediction challenge. The predictions were in poor agreement with experiment, and we conjectured that much of the error might derive from the force field, CHARMm with Vcharge charges. Repeating the calculations with other generalized force-fields led to no significant improvement, and we observed that the predicted affinities were highly sensitive to ...

  10. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    Science.gov (United States)

    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

  11. Predicting the Impact of Missense Mutations on Protein–Protein Binding Affinity

    OpenAIRE

    Li, Minghui; Petukh, Marharyta; Alexov, Emil; Panchenko, Anna R

    2014-01-01

    The crucial prerequisite for proper biological function is the protein’s ability to establish highly selective interactions with macromolecular partners. A missense mutation that alters the protein binding affinity may cause significant perturbations or complete abolishment of the function, potentially leading to diseases. The availability of computational methods to evaluate the impact of mutations on protein–protein binding is critical for a wide range of biomedical applications. Here, we r...

  12. Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation.

    Directory of Open Access Journals (Sweden)

    Luigi Capoferri

    Full Text Available 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.

  13. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

    Directory of Open Access Journals (Sweden)

    Lund Ole

    2007-07-01

    Full Text Available Abstract Background Antigen presenting cells (APCs sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC and three mouse H2-IA alleles. Results The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR, we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. Conclusion

  14. A Mixed QM/MM Scoring Function to Predict Protein-Ligand Binding Affinity

    OpenAIRE

    Hayik, Seth A.; Dunbrack, Roland; Merz, Kenneth M.

    2010-01-01

    Computational methods for predicting protein-ligand binding free energy continue to be popular as a potential cost-cutting method in the drug discovery process. However, accurate predictions are often difficult to make as estimates must be made for certain electronic and entropic terms in conventional force field based scoring functions. Mixed quantum mechanics/molecular mechanics (QM/MM) methods allow electronic effects for a small region of the protein to be calculated, treating the remaini...

  15. Predicting binding affinities of protein ligands from three-dimensional models: application to peptide binding to class I major histocompatibility proteins

    DEFF Research Database (Denmark)

    Rognan, D; Lauemoller, S L; Holm, A; Buus, S; Tschinke, V

    1999-01-01

    A simple and fast free energy scoring function (Fresno) has been developed to predict the binding free energy of peptides to class I major histocompatibility (MHC) proteins. It differs from existing scoring functions mainly by the explicit treatment of ligand desolvation and of unfavorable protein...... interactions were found to contribute the most to HLA-A0201-peptide interactions, whereas H-bonding predominates in H-2K(k) recognition. Both cross-validated models were afterward used to predict the binding affinity of a test set of 26 peptides to HLA-A0204 (an HLA allele closely related to HLA-A0201) and of...

  16. Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure-based modeling methods

    International Nuclear Information System (INIS)

    The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure–Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R2 = 0.55 and CCR = 0.76, respectively. In addition, for the first time a QSAR model was developed to predict binding affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R2 = 0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. - Highlights: • This is the largest curated dataset for ligand binding domain (LBD) of the THRβ. • We report the first QSAR model for antagonists of AF-2 domain of THRβ. • A combination of QSAR and docking enables prediction of both

  17. Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure-based modeling methods

    Energy Technology Data Exchange (ETDEWEB)

    Politi, Regina [Laboratory for Molecular Modeling, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina, Chapel Hill, NC 27599 (United States); Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599 (United States); Rusyn, Ivan, E-mail: iir@unc.edu [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599 (United States); Tropsha, Alexander, E-mail: alex_tropsha@unc.edu [Laboratory for Molecular Modeling, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina, Chapel Hill, NC 27599 (United States)

    2014-10-01

    The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure–Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R{sup 2} = 0.55 and CCR = 0.76, respectively. In addition, for the first time a QSAR model was developed to predict binding affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R{sup 2} = 0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. - Highlights: • This is the largest curated dataset for ligand binding domain (LBD) of the THRβ. • We report the first QSAR model for antagonists of AF-2 domain of THRβ. • A combination of QSAR and docking enables

  18. Integrating in silico and in vitro analysis of peptide binding affinity to HLA-Cw*0102: a bioinformatic approach to the prediction of new epitopes.

    Directory of Open Access Journals (Sweden)

    Valerie A Walshe

    Full Text Available BACKGROUND: Predictive models of peptide-Major Histocompatibility Complex (MHC binding affinity are important components of modern computational immunovaccinology. Here, we describe the development and deployment of a reliable peptide-binding prediction method for a previously poorly-characterized human MHC class I allele, HLA-Cw*0102. METHODOLOGY/FINDINGS: Using an in-house, flow cytometry-based MHC stabilization assay we generated novel peptide binding data, from which we derived a precise two-dimensional quantitative structure-activity relationship (2D-QSAR binding model. This allowed us to explore the peptide specificity of HLA-Cw*0102 molecule in detail. We used this model to design peptides optimized for HLA-Cw*0102-binding. Experimental analysis showed these peptides to have high binding affinities for the HLA-Cw*0102 molecule. As a functional validation of our approach, we also predicted HLA-Cw*0102-binding peptides within the HIV-1 genome, identifying a set of potent binding peptides. The most affine of these binding peptides was subsequently determined to be an epitope recognized in a subset of HLA-Cw*0102-positive individuals chronically infected with HIV-1. CONCLUSIONS/SIGNIFICANCE: A functionally-validated in silico-in vitro approach to the reliable and efficient prediction of peptide binding to a previously uncharacterized human MHC allele HLA-Cw*0102 was developed. This technique is generally applicable to all T cell epitope identification problems in immunology and vaccinology.

  19. Methods for Improving Aptamer Binding Affinity

    OpenAIRE

    Hijiri Hasegawa; Nasa Savory; Koichi Abe; Kazunori Ikebukuro

    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 a...

  20. Computational prediction of binding affinity for CYP1A2-ligand complexes using empirical free energy calculations

    DEFF Research Database (Denmark)

    Poongavanam, Vasanthanathan; Olsen, Lars; Jørgensen, Flemming Steen;

    2010-01-01

    , and methods based on statistical mechanics. In the present investigation, we started from an LIE model to predict the binding free energy of structurally diverse compounds of cytochrome P450 1A2 ligands, one of the important human metabolizing isoforms of the cytochrome P450 family. The data set...... includes both substrates and inhibitors. It appears that the electrostatic contribution to the binding free energy becomes negligible in this particular protein and a simple empirical model was derived, based on a training set of eight compounds. The root mean square error for the training set was 3.7 k...

  1. HADDOCK2P2I : A biophysical model for predicting the binding affinity of protein-protein interaction inhibitors

    NARCIS (Netherlands)

    Kastritis, Panagiotis L.; Garcia Lopes Maia Rodrigues, João; Bonvin, Alexandre M J J

    2014-01-01

    The HADDOCK score, a scoring function for both protein-protein and protein-nucleic acid modeling, has been successful in selecting near-native docking poses in a variety of cases, including those of the CAPRI blind prediction experiment. However, it has yet to be optimized for small molecules, and i

  2. Development of classification model and QSAR model for predicting binding affinity of endocrine disrupting chemicals to human sex hormone-binding globulin.

    Science.gov (United States)

    Liu, Huihui; Yang, Xianhai; Lu, Rui

    2016-08-01

    Disturbing the transport process is a crucial pathway for endocrine disrupting chemicals (EDCs) to disrupt endocrine function. However, this mechanism has not gotten enough attention, compared with that of hormone receptors and synthetase up to now, especially for the sex hormone transport process. In this study, we selected sex hormone-binding globulin (SHBG) and EDCs as a model system and the relative competing potency of a chemical with testosterone binding to SHBG (log RBA) as the endpoints, to develop classification models and quantitative structure-activity relationship (QSAR) models. With the classification model, a satisfactory model with nR09, nR10 and RDF155v as the most relevant variables was screened. Statistic results indicated that the model had the sensitivity, specificity, accuracy of 86.4%, 80.0%, 84.4% and 85.7%, 87.5%, 86.2% for the training set and validation set, respectively, highlighting a high classification performance of the model. With the QSAR model, a satisfactory model with statistical parameters, specifically, an adjusted determination coefficient (Radj(2)) of 0.810, a root mean square error (RMSE) of 0.616, a leave-one-out cross-validation squared correlation coefficient (QLOO(2)) of 0.777, a bootstrap method (QBOOT(2)) of 0.756, an external validation coefficient (Qext(2)) of 0.544 and a RMSEext of 0.859, were obtained, which implied satisfactory goodness of fit, robustness and predictive ability. The applicability domain of the current model covers a large number of structurally diverse chemicals, especially a few classes of nonsteroidal compounds. PMID:27156209

  3. Protein-ligand binding affinities from large-scale quantum mechanical simulations

    OpenAIRE

    Fox, Stephen J.

    2012-01-01

    The accurate prediction of protein-drug binding affinities is a major aim of computational drug optimisation and development. A quantitative measure of binding affinity is provided by the free energy of binding, and such calculations typically require extensive configurational sampling of entities such as proteins with thousands of atoms. Current binding free energy methods use force fields to perform the configurational sampling and to compute interaction energies. Due to the empirical natur...

  4. In Silico Prediction of Estrogen Receptor Subtype Binding Affinity and Selectivity Using Statistical Methods and Molecular Docking with 2-Arylnaphthalenes and 2-Arylquinolines

    Directory of Open Access Journals (Sweden)

    Yonghua Wang

    2010-09-01

    Full Text Available Over the years development of selective estrogen receptor (ER ligands has been of great concern to researchers involved in the chemistry and pharmacology of anticancer drugs, resulting in numerous synthesized selective ER subtype inhibitors. In this work, a data set of 82 ER ligands with ERα and ERβ inhibitory activities was built, and quantitative structure-activity relationship (QSAR methods based on the two linear (multiple linear regression, MLR, partial least squares regression, PLSR and a nonlinear statistical method (Bayesian regularized neural network, BRNN were applied to investigate the potential relationship of molecular structural features related to the activity and selectivity of these ligands. For ERα and ERβ, the performances of the MLR and PLSR models are superior to the BRNN model, giving more reasonable statistical properties (ERα: for MLR, Rtr2 = 0.72, Qte2 = 0.63; for PLSR, Rtr2 = 0.92, Qte2 = 0.84. ERβ: for MLR, Rtr2 = 0.75, Qte2 = 0.75; for PLSR, Rtr2 = 0.98, Qte2 = 0.80. The MLR method is also more powerful than other two methods for generating the subtype selectivity models, resulting in Rtr2 = 0.74 and Qte2 = 0.80. In addition, the molecular docking method was also used to explore the possible binding modes of the ligands and a relationship between the 3D-binding modes and the 2D-molecular structural features of ligands was further explored. The results show that the binding affinity strength for both ERα and ERβ is more correlated with the atom fragment type, polarity, electronegativites and hydrophobicity. The substitutent in position 8 of the naphthalene or the quinoline plane and the space orientation of these two planes contribute the most to the subtype selectivity on the basis of similar hydrogen bond interactions between binding ligands and both ER subtypes. The QSAR models built together with the docking procedure should be of great advantage for screening and designing ER ligands with improved affinity

  5. Candidate SNP Markers of Chronopathologies Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

    Science.gov (United States)

    Ponomarenko, Petr; Rasskazov, Dmitry; Suslov, Valentin; Sharypova, Ekaterina; Savinkova, Ludmila; Podkolodnaya, Olga; Podkolodny, Nikolay L.; Tverdokhleb, Natalya N.; Chadaeva, Irina; Kolchanov, Nikolay

    2016-01-01

    Variations in human genome (e.g., single nucleotide polymorphisms, SNPs) may be associated with hereditary diseases, their complications, comorbidities, and drug responses. Using Web service SNP_TATA_Comparator presented in our previous paper, here we analyzed immediate surroundings of known SNP markers of diseases and identified several candidate SNP markers that can significantly change the affinity of TATA-binding protein for human gene promoters, with circadian consequences. For example, rs572527200 may be related to asthma, where symptoms are circadian (worse at night), and rs367732974 may be associated with heart attacks that are characterized by a circadian preference (early morning). By the same method, we analyzed the 90 bp proximal promoter region of each protein-coding transcript of each human gene of the circadian clock core. This analysis yielded 53 candidate SNP markers, such as rs181985043 (susceptibility to acute Q fever in male patients), rs192518038 (higher risk of a heart attack in patients with diabetes), and rs374778785 (emphysema and lung cancer in smokers). If they are properly validated according to clinical standards, these candidate SNP markers may turn out to be useful for physicians (to select optimal treatment for each patient) and for the general population (to choose a lifestyle preventing possible circadian complications of diseases).

  6. Prediction of binding modes and affinities of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamide inhibitors to the carbonic anhydrase receptor by docking and ONIOM calculations.

    Science.gov (United States)

    Samanta, Pabitra Narayan; Das, Kalyan Kumar

    2016-01-01

    Inhibition activities of a series of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamides against the human carbonic anhydrase II (HCAII) enzyme have been explored by employing molecular docking and hybrid QM/MM methods. The docking protocol has been employed to assess the best pose of each ligand in the active site cavity of the enzyme, and probe the interactions with the amino acid residues. The docking calculations reveal that the inhibitor binds to the catalytic Zn(2+) site through the deprotonated sulfonamide nitrogen atom by making several hydrophobic and hydrogen bond interactions with the side chain residues depending on the substituted moiety. A cross-docking approach has been adopted prior to the hybrid QM/MM calculation to validate the docked poses. A correlation between the experimental dissociation constants and the docked free energies for the enzyme-inhibitor complexes has been established. Two-layered ONIOM calculations based on QM/MM approach have been performed to evaluate the binding efficacy of the inhibitors. The inhibitor potency has been predicted from the computed binding energies after taking into account of the electronic phenomena associated with enzyme-inhibitor interactions. Both the hybrid (B3LYP) and meta-hybrid (M06-2X) functionals are used for the description of the QM region. To improve the correlation between the experimental biological activity and the theoretical results, a three-layered ONIOM calculation has been carried out and verified for some of the selected inhibitors. The charge transfer stabilization energies are calculated via natural bond orbital analysis to recognize the donor-acceptor interaction in the binding pocket of the enzyme. The nature of binding between the inhibitors and HCAII active site is further analyzed from the electron density distribution maps. PMID:26619075

  7. Calculation of Host-Guest Binding Affinities Using a Quantum-Mechanical Energy Model

    OpenAIRE

    Muddana, Hari S.; Gilson, Michael K.

    2012-01-01

    The prediction of protein-ligand binding affinities is of central interest in computer-aided drug discovery, but it is still difficult to achieve a high degree of accuracy. Recent studies suggesting that available force fields may be a key source of error motivate the present study, which reports the first mining minima (M2) binding affinity calculations based on a quantum mechanical energy model, rather than an empirical force field. We apply a semi-empirical quantum-mechanical energy functi...

  8. Tetrapyrrole binding affinity of the murine and human p22HBP heme-binding proteins.

    Science.gov (United States)

    Micaelo, Nuno M; Macedo, Anjos L; Goodfellow, Brian J; Félix, Vítor

    2010-11-01

    We present the first systematic molecular modeling study of the binding properties of murine (mHBP) and human (hHBP) p22HBP protein (heme-binding protein) with four tetrapyrrole ring systems belonging to the heme biosynthetic pathway: iron protoporphyrin IX (HEMIN), protoporphyrin IX (PPIX), coproporphyrin III (CPIII), coproporphyrin I (CPI). The relative binding affinities predicted by our computational study were found to be similar to those observed experimentally, providing a first rational structural analysis of the molecular recognition mechanism, by p22HBP, toward a number of different tetrapyrrole ligands. To probe the structure of these p22HBP protein complexes, docking, molecular dynamics and MM-PBSA methodologies supported by experimental NMR ring current shift data have been employed. The tetrapyrroles studied were found to bind murine p22HBP with the following binding affinity order: HEMIN> PPIX> CPIII> CPI, which ranged from -22.2 to -6.1 kcal/mol. In general, the protein-tetrapyrrole complexes are stabilized by non-bonded interactions between the tetrapyrrole propionate groups and basic residues of the protein, and by the preferential solvation of the complex compared to the unbound components. PMID:20800521

  9. Relative Binding Affinities of Monolignols to Horseradish Peroxidase.

    Science.gov (United States)

    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

  10. Affinity purification of proteins binding to GST fusion proteins.

    Science.gov (United States)

    Swaffield, J C; Johnston, S A

    2001-05-01

    This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding. PMID:18265191

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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-17β to the four rainbow trout ER isoforms with that of three known environmental estrogens 17α-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 ERα subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17β, bisphenol A binds less strongly to all four receptors, 17α-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the α 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.

  13. Agonist binding to high-affinity dopamine sites

    Energy Technology Data Exchange (ETDEWEB)

    Tedesco, J.L.

    1985-01-01

    The authors have characterized the dopamine D/sub 3/ site and its binding requirements. The dopamine D/sub 3/ site in calf caudate crude homogenate has a site density of 214-230 fmoles/mg. protein by both /sup 3/H-apomorphine (/sup 3/H-AOP) and /sup 3/H-dopamine (/sup 3/H-DA) Scatchard analysis of specific binding (SB). Stereospecific subsets of /sup 3/H-APO and /sup 3/H-DA sites were defined by the use of agonist and antagonist enantiomer-pairs as a rigorous test for D/sub 3/ site heterogeneity. IC/sub 50/ values for both /sup 3/H-APO and /sup 3/H-DA SB sites were assessed for 55 agonist ligands and an excellent correlation was obtained. The authors conclude that both /sup 3/H-ligands label the same D/sub 3/ site. The D/sub 3/ site affinities of 105 dopamine-agonist ligands, in particular 2-aminotetralins,, aporphines and flexible dopamine analogues were measured. Low D/sub 3/-site affinities of N-quaternary analogues confirm the need for a lone pair. Subadditivity of substituents' effects in semi-flexible DA analogues confirms their postulate that sidechain conformation is the critical determinant of affinity. They conclude that there are at least two high-affinity ligand conformations of the DA sidechain pharmacophore. These binding requirements are presented as two interface-Geometry tetrahedral models of the double H-bond interface between the D/sub 3/ site and the ideal ligand.

  14. Detecting and correcting the binding-affinity bias in ChIP-seq data using inter-species information

    OpenAIRE

    Nettling, Martin; Treutler, Hendrik; Cerquides, Jesus; Grosse, Ivo

    2016-01-01

    Background Transcriptional gene regulation is a fundamental process in nature, and the experimental and computational investigation of DNA binding motifs and their binding sites is a prerequisite for elucidating this process. ChIP-seq has become the major technology to uncover genomic regions containing those binding sites, but motifs predicted by traditional computational approaches using these data are distorted by a ubiquitous binding-affinity bias. Here, we present an approach for detecti...

  15. Determinants of benzodiazepine brain uptake: lipophilicity versus binding affinity.

    Science.gov (United States)

    Arendt, R M; Greenblatt, D J; Liebisch, D C; Luu, M D; Paul, S M

    1987-01-01

    Factors influencing brain uptake of benzodiazepine derivatives were evaluated in adult Sprague Dawley rats (n = 8-10 per drug). Animals received single intraperitoneal doses of alprazolam, triazolam, lorazepam, flunitrazepam, diazepam, midazolam, desmethyldiazepam, or clobazam. Concentrations of each drug (and metabolites) in whole brain and serum 1 h after dosage were determined by gas chromatography. Serum free fraction was measured by equilibrium dialysis. In vitro binding affinity (apparent Ki) of each compound was estimated based on displacement of tritiated flunitrazepam in washed membrane preparations from rat cerebral cortex. Lipid solubility of each benzodiazepine was estimated using the reverse-phase liquid chromatographic (HPLC) retention index at physiologic pH. There was no significant relation between brain:total serum concentration ratio and either HPLC retention (r = 0.18) or binding Ki (r = -0.34). Correction of uptake ratios for free as opposed to total serum concentration yielded a highly significant correlation with HPLC retention (r = 0.78, P less than 0.005). However, even the corrected ratio was not correlated with binding Ki (r = -0.22). Thus a benzodiazepine's capacity to diffuse from systemic blood into brain tissue is much more closely associated with the physicochemical property of lipid solubility than with specific affinity. Unbound rather than total serum or plasma concentration most accurately reflects the quantity of drug available for diffusion. PMID:2888155

  16. Binding Affinity and Capacity for the Uremic Toxin Indoxyl Sulfate

    Directory of Open Access Journals (Sweden)

    Eric Devine

    2014-01-01

    Full Text Available Protein binding prevents uremic toxins from removal by conventional extracorporeal therapies leading to accumulation in maintenance dialysis patients. Weakening of the protein binding may enhance the dialytic elimination of these toxins. In ultrafiltration and equilibrium dialysis experiments, different measures to modify the plasma binding affinity and capacity were tested: (i, increasing the sodium chloride (NaCl concentration to achieve a higher ionic strength; (ii, increasing the temperature; and (iii, dilution. The effects on the dissociation constant KD and the protein bound fraction of the prototypical uremic toxin indoxyl sulfate (IS in plasma of healthy and uremic individuals were studied. Binding of IS corresponded to one site binding in normal plasma. KD increased linearly with the NaCl concentration between 0.15 (KD = 13.2 ± 3.7 µM and 0.75 M (KD = 56.2 ± 2.0 µM. Plasma dilution further reduced the protein bound toxin fraction by lowering the protein binding capacity of the plasma. Higher temperatures also decreased the protein bound fraction of IS in human plasma. Increasing the NaCl concentration was effective to weaken the binding of IS also in uremic plasma: the protein bound fraction decreased from 89% ± 3% to 81% ± 3% at 0.15 and 0.75 M NaCl, respectively. Dilution and increasing the ionic strength and temperature enhance the free fraction of IS allowing better removal of the substance during dialysis. Applied during clinical dialysis, this may have beneficial effects on the long-term outcome of maintenance dialysis patients.

  17. Free energy calculations to estimate ligand-binding affinities in structure-based drug design.

    Science.gov (United States)

    Reddy, M Rami; Reddy, C Ravikumar; Rathore, R S; Erion, Mark D; Aparoy, P; Reddy, R Nageswara; Reddanna, P

    2014-01-01

    Post-genomic era has led to the discovery of several new targets posing challenges for structure-based drug design efforts to identify lead compounds. Multiple computational methodologies exist to predict the high ranking hit/lead compounds. Among them, free energy methods provide the most accurate estimate of predicted binding affinity. Pathway-based Free Energy Perturbation (FEP), Thermodynamic Integration (TI) and Slow Growth (SG) as well as less rigorous end-point methods such as Linear interaction energy (LIE), Molecular Mechanics-Poisson Boltzmann./Generalized Born Surface Area (MM-PBSA/GBSA) and λ-dynamics have been applied to a variety of biologically relevant problems. The recent advances in free energy methods and their applications including the prediction of protein-ligand binding affinity for some of the important drug targets have been elaborated. Results using a recently developed Quantum Mechanics (QM)/Molecular Mechanics (MM) based Free Energy Perturbation (FEP) method, which has the potential to provide a very accurate estimation of binding affinities to date has been discussed. A case study for the optimization of inhibitors for the fructose 1,6- bisphosphatase inhibitors has been described. PMID:23947646

  18. High-throughput analysis of protein-DNA binding affinity.

    Science.gov (United States)

    Franco-Zorrilla, José M; Solano, Roberto

    2014-01-01

    Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

  19. THE RECEPTOR BINDING AFFINITIES, ANTIPROGESTERONE AND ANTIGLUCOCORTICOID ACTIVITIES OF MIFEPRISTONE AND LILOPRISTONE

    Institute of Scientific and Technical Information of China (English)

    LIUYong-Qiang; WUXi-Rui

    1989-01-01

    With radioligand binding assays, the receptor binding affmities of mifepristone and lilopristone to the rabbit uterus cytosol progesterone receptor and the rat fiver cytosol glucocorticoid receptor have been measured. The relative binding affinities ( RBA ) of

  20. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

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

    Directory of Open Access Journals (Sweden)

    Graham S Baldwin

    Full Text Available 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.

  2. PBSA_E: A PBSA-Based Free Energy Estimator for Protein-Ligand Binding Affinity.

    Science.gov (United States)

    Liu, Xiao; Liu, Jinfeng; Zhu, Tong; Zhang, Lujia; He, Xiao; Zhang, John Z H

    2016-05-23

    Improving the accuracy of scoring functions for estimating protein-ligand binding affinity is of significant interest as well as practical utility in drug discovery. In this work, PBSA_E, a new free energy estimator based on the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) descriptors, has been developed. This free energy estimator was optimized using high-quality experimental data from a training set consisting of 145 protein-ligand complexes. The method was validated on two separate test sets containing 121 and 130 complexes. Comparison of the binding affinities predicted using the present method with those obtained using three popular scoring functions, i.e., GlideXP, GlideSP, and SYBYL_F, demonstrated that the PBSA_E method is more accurate. This new energy estimator requires a MM/PBSA calculation of the protein-ligand binding energy for a single complex configuration, which is typically obtained by optimizing the crystal structure. The present study shows that PBSA_E has the potential to become a robust tool for more reliable estimation of protein-ligand binding affinity in structure-based drug design. PMID:27088302

  3. Modelling the binding affinity of steroids to zebrafish sex hormone-binding globulin.

    Science.gov (United States)

    Saxena, A K; Devillers, J; Pery, A R R; Beaudouin, R; Balaramnavar, V M; Ahmed, S

    2014-01-01

    The circulating endogenous steroids are transported in the bloodstream. These are bound to a highly specific sex hormone-binding globulin (SHBG) and in lower affinity to proteins such as the corticosteroid-binding protein and albumin in vertebrates, including fish. It is generally believed that the glycoprotein SHBG protects these steroids from rapid metabolic degradation and thus intervenes in its availability at the target tissues. Endocrine disrupters binding to SHBG affect the normal activity of natural steroids. Since xenobiotics are primarily released in the aquatic environment, there is a need to evaluate the binding affinity of xenosteroid mimics on fish SHBG, especially in zebrafish (Danio rerio), a small freshwater fish originating in India and widely employed in ecotoxicology, toxicology, and genetics. In this context, a zebrafish SHBG (zfSHBG) homology model was developed using the human SHBG (hSHBG) receptor structure as template. It was shown that interactions with amino acids Ser-36, Asp-59 and Thr-54 were important for binding affinity. A ligand-based pharmacophore model was also developed for both zfSHBG and hSHBG inhibitors that differentiated binders from non-binders, but also demonstrated structural requirements for zfSHBG and hSHBG ligands. The study provides insights into the mechanism of action of endocrine disruptors in zebrafish as well as providing a useful tool for identifying anthropogenic compounds inhibiting zfSHBG. PMID:24874994

  4. Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays

    DEFF Research Database (Denmark)

    Harndahl, Mikkel; Justesen, Sune Frederik Lamdahl; Lamberth, Kasper;

    2009-01-01

    The Human MHC Project aims at large-scale description of peptide-HLA binding to a wide range of HLA molecules covering all populations of the world and the accompanying generation of bioinformatics tools capable of predicting binding of any given peptide to any given HLA molecule. Here, the authors...... present a homogenous, proximity-based assay for detection of peptide binding to HLA class I molecules. It uses a conformation-dependent anti-HLA class I antibody, W6/32, as one tag and a biotinylated recombinant HLA class I molecule as the other tag, and a proximity-based signal is generated through the...... luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen (TM)). Compared with an enzyme-linked immunosorbent assay-based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range...

  5. Synthesis and binding affinity of an iodinated juvenile hormone

    International Nuclear Information System (INIS)

    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 3H-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 [125I]12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added 125I-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 [125I]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

  6. Influences of hydrocarbon linkers on the receptor binding affinities of gonadotropin-releasing hormone peptides

    OpenAIRE

    Guo, Haixun; Hathaway, Helen; Royce, Melanie E.; Prossnitz, Eric R.; Miao, Yubin

    2013-01-01

    Three new DOTA-conjugated GnRH peptides with various hydrocarbon linkers were synthesized to evaluate the influences of the linkers on their receptor binding affinities. The hydrocarbon linker displayed a profound impact on the receptor binding affinities of DOTA-conjugated GnRH peptides. The Aun linker was better than Gaba, Ahx and Aoc linkers in retaining strong receptor binding affinity of the GnRH peptide. DOTA-Aun-(D-Lys6-GnRH) displayed 22.8 nM GnRH receptor binding affinity. 111In-DOTA...

  7. Metal binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana.

    Science.gov (United States)

    Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Giorgetti, Alejandro; Dominici, Paola; Astegno, Alessandra

    2016-08-01

    In addition to the well-known Ca(2+) sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca(2+) and Mg(2+) binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca(2+) ion with micromolar affinity (Kd ∼ 12 µM) and the presence of 10 mM Mg(2+) decreases the Ca(2+) affinity by ∼5-fold. Although binding of Ca(2+) to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca(2+) sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role. PMID:27124620

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

    Science.gov (United States)

    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

  9. Inhibition of aggregation of amyloid peptides by beta-sheet breaker peptides and their binding affinity.

    Science.gov (United States)

    Viet, Man Hoang; Ngo, Son Tung; Lam, Nguyen Sy; Li, Mai Suan

    2011-06-01

    The effects of beta-sheet breaker peptides KLVFF and LPFFD on the oligomerization of amyloid peptides were studied by all-atom simulations. It was found that LPFFD interferes the aggregation of Aβ(16-22) peptides to a greater extent than does KLVFF. Using the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method, we found that the former binds more strongly to Aβ(16-22). Therefore, by simulations, we have clarified the relationship between aggregation rates and binding affinity: the stronger the ligand binding, the slower the oligomerization process. The binding affinity of pentapeptides to full-length peptide Aβ(1-40) and its mature fibrils has been considered using the Autodock and MM-PBSA methods. The hydrophobic interaction between ligands and receptors plays a more important role for association than does hydrogen bonding. The influence of beta-sheet breaker peptides on the secondary structures of monomer Aβ(1-40) was studied in detail, and it turns out that, in their presence, the total beta-sheet content can be enhanced. However, the aggregation can be slowed because the beta-content is reduced in fibril-prone regions. Both pentapeptides strongly bind to monomer Aβ(1-40), as well as to mature fibrils, but KLVFF displays a lower binding affinity than LPFFD. Our findings are in accord with earlier experiments that both of these peptides can serve as prominent inhibitors. In addition, we predict that LPFFD inhibits/degrades the fibrillogenesis of full-length amyloid peptides better than KLVFF. This is probably related to a difference in their total hydrophobicities in that the higher the hydrophobicity, the lower the inhibitory capacity. The GROMOS96 43a1 force field with explicit water and the force field proposed by Morris et al. (Morris et al. J. Comput. Chem. 1998, 19, 1639 ) were employed for all-atom molecular dynamics simulations and Autodock experiments, respectively. PMID:21563780

  10. Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

    International Nuclear Information System (INIS)

    Research highlights: → PD153035 is a DNA intercalator and intercalation occurs only under very low salt concentration. → The minimum distance between adjacent bound PD153035 ∼ 11 bp. → Binding affinity constant for PD153035 is 1.18(±0.09) x 104 (1/M). → The change of binding free energy of PD153035-DNA interaction is -5.49 kcal mol-1 at 23 ± 0.5 oC. -- Abstract: Accurately predicting binding affinity constant (KA) is highly required to determine the binding energetics of the driving forces in drug-DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining KA for PD153035, where KA is determined from the changes in B-form contour length (L) of PD153035-DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1 mM sodium cacodylate was exhibited. Furthermore, our results showed that KA = 1.18(±0.09) x 104 (1/M) at 23 ± 0.5 oC and the minimum distance between adjacent bound PD153035 ∼ 11 bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.

  11. A structure-based model for predicting serum albumin binding.

    Directory of Open Access Journals (Sweden)

    Katrina W Lexa

    Full Text Available One of the many factors involved in determining the distribution and metabolism of a compound is the strength of its binding to human serum albumin. While experimental and QSAR approaches for determining binding to albumin exist, various factors limit their ability to provide accurate binding affinity for novel compounds. Thus, to complement the existing tools, we have developed a structure-based model of serum albumin binding. Our approach for predicting binding incorporated the inherent flexibility and promiscuity known to exist for albumin. We found that a weighted combination of the predicted logP and docking score most accurately distinguished between binders and nonbinders. This model was successfully used to predict serum albumin binding in a large test set of therapeutics that had experimental binding data.

  12. Improving the scoring of protein-ligand binding affinity by including the effects of structural water and electronic polarization.

    Science.gov (United States)

    Liu, Jinfeng; He, Xiao; Zhang, John Z H

    2013-06-24

    Docking programs that use scoring functions to estimate binding affinities of small molecules to biological targets are widely applied in drug design and drug screening with partial success. But accurate and efficient scoring functions for protein-ligand binding affinity still present a grand challenge to computational chemists. In this study, the polarized protein-specific charge model (PPC) is incorporated into the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method to rescore the binding poses of some protein-ligand complexes, for which docking programs, such as Autodock, could not predict their binding modes correctly. Different sampling techniques (single minimized conformation and multiple molecular dynamics (MD) snapshots) are used to test the performance of MM/PBSA combined with the PPC model. Our results show the availability and effectiveness of this approach in correctly ranking the binding poses. More importantly, the bridging water molecules are found to play an important role in correctly determining the protein-ligand binding modes. Explicitly including these bridging water molecules in MM/PBSA calculations improves the prediction accuracy significantly. Our study sheds light on the importance of both bridging water molecules and the electronic polarization in the development of more reliable scoring functions for predicting molecular docking and protein-ligand binding affinity. PMID:23651068

  13. APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

    OpenAIRE

    Sviatenko, О.; Gorbatiuk, O.; Vasylchenko, О.

    2014-01-01

    Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding speci...

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

    Science.gov (United States)

    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.

  15. Peptide binding predictions for HLA DR, DP and DQ molecules

    DEFF Research Database (Denmark)

    Wang, P.; Sidney, J.; Kim, Y.;

    2010-01-01

    BACKGROUND: MHC class II binding predictions are widely used to identify epitope candidates in infectious agents, allergens, cancer and autoantigens. The vast majority of prediction algorithms for human MHC class II to date have targeted HLA molecules encoded in the DR locus. This reflects a...... significant gap in knowledge as HLA DP and DQ molecules are presumably equally important, and have only been studied less because they are more difficult to handle experimentally. RESULTS: In this study, we aimed to narrow this gap by providing a large scale dataset of over 17,000 HLA-peptide binding...... affinities for a set of 11 HLA DP and DQ alleles. We also expanded our dataset for HLA DR alleles resulting in a total of 40,000 MHC class II binding affinities covering 26 allelic variants. Utilizing this dataset, we generated prediction tools utilizing several machine learning algorithms and evaluated...

  16. Immunoglobulin G preparation from plasma samples and analysis of its affinity kinetic binding to peptide hormones

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Romain Legrand, Kuniko Takagi & Sergueï Fetissov ### Abstract Circulating peptide hormones such as ghrelin physiologically bind plasma immunoglobulins (Ig) which protect hormone from degradation and modulate its biological activity depending on affinity of hormone / IgG binding. Because the IgG set of each individual is unique, measuring affinity kinetics of human or animal plasma IgG binding to a specific peptide hormone may provide useful information towards understandi...

  17. Isotope-coded ATP Probe for Quantitative Affinity Profiling of ATP-binding Proteins

    OpenAIRE

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2013-01-01

    ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale. False-positive identification of ATP-binding ...

  18. Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities

    OpenAIRE

    Ader, Lauren; Jones, David N. M.; Lin, Hai

    2010-01-01

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as cr...

  19. Synthesis and receptor binding affinity of new selective GluR5 ligands

    DEFF Research Database (Denmark)

    Bunch, L; Johansen, T H; Bräuner-Osborne, Hans;

    2001-01-01

    Two hybrid analogues of the kainic acid receptor agonists, 2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA) and (2S,4R)-4-methylglutamic acid ((2S,4R)-4-Me-Glu), were designed, synthesized, and characterized in radioligand binding assays using cloned ionotropic and metabotropi.......0 and 2.0 microM. respectively. Their affinities in the [3H]AMPA binding assay on native cortical receptors were shown to correlate with their GluR2 affinity rather than their GluR5 affinity. No affinity for GluR6 was detected (IC50 > 100 microM)....

  20. Production and Characterization of Desmalonichrome Relative Binding Affinity for Uranyl Ions in Relation to other Siderophores

    Energy Technology Data Exchange (ETDEWEB)

    Mo, Kai-For; Dai, Ziyu; Wunschel, David S.

    2016-05-27

    Siderophores are Fe binding secondary metabolites that have been investigated for their uranium binding properties. Much of the previous work has focused on characterizing hydroxamate types of siderophores, such as desferrioxamine B, for their uranyl binding affinity. Carboxylate forms of these metabolites hold potential to be more efficient chelators of uranyl, yet they have not been widely studied and are more difficult to obtain. Desmalonichrome is a carboxylate siderophore which is not commercially available and so was obtained from the ascomycete fungus Fusarium oxysporum cultivated under Fe depleted conditions. The relative affinity for uranyl binding of desmalonichrome was investigated using a competitive analysis of binding affinities between uranyl acetate and different concentrations of iron(III) chloride using electrospray ionization mass spectrometry (ESI-MS). In addition to desmalonichrome, three other siderophores, including two hydroxamates (desferrioxamine B and desferrichrome) and one carboxylate (desferrichrome A) were studied to understand their relative affinities for the uranyl ion at two pH values. The binding affinities of hydroxymate siderophores to uranyl ion were found to decrease to a greater degree at lower pH as the concentration of Fe (III) ion increases. On the other hand, lowering pH has little impact on the binding affinities between carboxylate siderophores and uranyl ion. Desmalonichrome was shown to have the greatest relative affinity for uranyl at any pH and Fe(III) concentration. These results suggest that acidic functional groups in the ligands are critical for strong chelation with uranium at lower pH.

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

    Science.gov (United States)

    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

  2. Parameterization of an effective potential for protein-ligand binding from host-guest affinity data.

    Science.gov (United States)

    Wickstrom, Lauren; Deng, Nanjie; He, Peng; Mentes, Ahmet; Nguyen, Crystal; Gilson, Michael K; Kurtzman, Tom; Gallicchio, Emilio; Levy, Ronald M

    2016-01-01

    Force field accuracy is still one of the "stalemates" in biomolecular modeling. Model systems with high quality experimental data are valuable instruments for the validation and improvement of effective potentials. With respect to protein-ligand binding, organic host-guest complexes have long served as models for both experimental and computational studies because of the abundance of binding affinity data available for such systems. Binding affinity data collected for cyclodextrin (CD) inclusion complexes, a popular model for molecular recognition, is potentially a more reliable resource for tuning energy parameters than hydration free energy measurements. Convergence of binding free energy calculations on CD host-guest systems can also be obtained rapidly, thus offering the opportunity to assess the robustness of these parameters. In this work, we demonstrate how implicit solvent parameters can be developed using binding affinity experimental data and the binding energy distribution analysis method (BEDAM) and validated using the Grid Inhomogeneous Solvation Theory analysis. These new solvation parameters were used to study protein-ligand binding in two drug targets against the HIV-1 virus and improved the agreement between the calculated and the experimental binding affinities. This work illustrates how benchmark sets of high quality experimental binding affinity data and physics-based binding free energy models can be used to evaluate and optimize force fields for protein-ligand systems. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26256816

  3. High affinity binding of [3H]cocaine to rat liver microsomes

    International Nuclear Information System (INIS)

    ]3H]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 [3H]cocaine binding. On the other hand, chronic administration of cocaine reduced [3H]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 [3H]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 [3H]cocaine binding to liver with a different rank order of potency than their displacement of [3H]cocaine binding to striatum. This high affinity [3H]cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  4. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

    OpenAIRE

    Turk, B.; Stoka, V.; Björk, I.; Boudier, C.; Johansson, G.; Dolenc, I.; Colic, A.; Bieth, J. G.; Turk, V.

    1995-01-01

    Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants k...

  5. Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus

    NARCIS (Netherlands)

    De Kloet, E R; Veldhuis, H D; Wagenaars, J L; Bergink, E W

    1984-01-01

    In cytosol of the hippocampus corticosterone displays highest affinity for the sites that remain available for binding in the presence of excess RU 26988, which is shown to be a "pure" glucocorticoid. A rather high affinity (greater than or equal to 25%) was found for 11 beta-hydroxyprogesterone, 21

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

    Science.gov (United States)

    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)

  7. Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities.

    Science.gov (United States)

    Ribeiro, Teresa; Santos-Silva, Teresa; Alves, Victor D; Dias, Fernando M V; Luís, Ana S; Prates, José A M; Ferreira, Luís M A; Romão, Maria J; Fontes, Carlos M G A

    2010-10-01

    Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as alpha, beta and gamma. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the gamma sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of beta and gamma sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities. PMID:20637315

  8. Preliminary study of the metal binding site of an anti-DTPA-indium antibody by equilibrium binding immunoassays and immobilized metal ion affinity chromatography.

    Science.gov (United States)

    Boden, V; Colin, C; Barbet, J; Le Doussal, J M; Vijayalakshmi, M

    1995-01-01

    Creating metal coordination sites by modifying an existing enzyme or by eliciting antibodies against metal chelate haptens is of great interest in biotechnology to create enzyme catalysts with novel specificities. Here, we investigate the metal binding potential of a monoclonal antibody raised against a DTPA-In(III) hapten (mAb 734). We study its relative binding efficiency to metals of biological relevance by equilibrium binding immunoassays and immobilized metal ion affinity chromatography, two approaches which can give complementary information regarding composition and/or structure of the metal binding site(s). Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II) binding was compared to In(III). All of them were shown to displace indium, but their affinity for mAb 734 decreased by 100-fold compared to indium. Competitive metal binding immunoassays between Zn(II) and In(III) revealed an unusual behavior by Zn(II) which remains to be explained. Moreover, IMAC allowed us to predict the metal binding amino acids involved in the antibody paratope. The antibody metal binding site was shown to contain at least two histidine residues in a cluster, and the presence of aspartic and glutamic acid as well as cysteine residues could not be excluded. Thus, simple competition studies allows us to obtain some partial information on the metal binding structural features of this anti-metal chelate antibody and to guide our screening of its catalytic potential. PMID:7578356

  9. Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.

    Science.gov (United States)

    Fielding, Lee; Rutherford, Samantha; Fletcher, Dan

    2005-06-01

    The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems. PMID:15816062

  10. Candidate SNP Markers of Gender-Biased Autoimmune Complications of Monogenic Diseases Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

    Science.gov (United States)

    Ponomarenko, Mikhail P.; Arkova, Olga; Rasskazov, Dmitry; Ponomarenko, Petr; Savinkova, Ludmila; Kolchanov, Nikolay

    2016-01-01

    Some variations of human genome [for example, single nucleotide polymorphisms (SNPs)] are markers of hereditary diseases and drug responses. Analysis of them can help to improve treatment. Computer-based analysis of millions of SNPs in the 1000 Genomes project makes a search for SNP markers more targeted. Here, we combined two computer-based approaches: DNA sequence analysis and keyword search in databases. In the binding sites for TATA-binding protein (TBP) in human gene promoters, we found candidate SNP markers of gender-biased autoimmune diseases, including rs1143627 [cachexia in rheumatoid arthritis (double prevalence among women)]; rs11557611 [demyelinating diseases (thrice more prevalent among young white women than among non-white individuals)]; rs17231520 and rs569033466 [both: atherosclerosis comorbid with related diseases (double prevalence among women)]; rs563763767 [Hughes syndrome-related thrombosis (lethal during pregnancy)]; rs2814778 [autoimmune diseases (excluding multiple sclerosis and rheumatoid arthritis) underlying hypergammaglobulinemia in women]; rs72661131 and rs562962093 (both: preterm delivery in pregnant diabetic women); and rs35518301, rs34166473, rs34500389, rs33981098, rs33980857, rs397509430, rs34598529, rs33931746, rs281864525, and rs63750953 (all: autoimmune diseases underlying hypergammaglobulinemia in women). Validation of these predicted candidate SNP markers using the clinical standards may advance personalized medicine. PMID:27092142

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

    International Nuclear Information System (INIS)

    The plasmalemmal serotonin transporter uses transmembrane gradients of Na+, Cl- and K+ to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na+. 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+ and Cl-, 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+ binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl-. Cl- enhances the transporters affinity for imipramine, as well as for Na+. At concentrations in the range of its KM 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+-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 [3H]imipramine binding and [3H]serotonin transport

  12. Protein-protein binding affinities calculated using the LIE method

    OpenAIRE

    Andberg, Tor Arne Heim

    2011-01-01

    Absolute binding free energies for the third domain of the turkey ovomucoid inhibitor in complex with Streptomyces griseus proteinase B and porcine pancreatic elastase has been calculated using the linear interaction energy method.

  13. The high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter.

    Science.gov (United States)

    Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H; Ecker, Gerhard F; Freissmuth, Michael; Sucic, Sonja

    2010-12-01

    The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuT(Aa)) has been used as a model for mammalian Na(+)/Cl(-)-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuT(Aa) liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERT(Y95F) mutation, which greatly reduced the affinity for [(3)H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [(3)H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP(+), paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants. PMID:20829432

  14. Production and Characterization of Desmalonichrome Relative Binding Affinity for Uranyl Ions in Relation to Other Siderophores.

    Science.gov (United States)

    Mo, Kai-For; Dai, Ziyu; Wunschel, David S

    2016-06-24

    Siderophores are iron (Fe)-binding secondary metabolites that have been investigated for their uranium-binding properties. Previous work has focused on characterizing hydroxamate types of siderophores, such as desferrioxamine B, for their uranyl (UO2)-binding affinity. Carboxylate forms of these metabolites hold potential to be more efficient chelators of UO2, yet they have not been widely studied. Desmalonichrome is a carboxylate siderophore that is not commercially available and so was obtained from the fungus Fusarium oxysporum cultivated under Fe-depleted conditions. The relative affinity for UO2 binding of desmalonichrome was investigated using a competitive analysis of binding affinities between UO2 acetate and different concentrations of Fe(III) chloride using electrospray ionization mass spectrometry. In addition to desmalonichrome, three other siderophores, including two hydroxamates (desferrioxamine B and desferrichrome) and one carboxylate (desferrichrome A), were studied to understand their relative affinities for the UO2(2+) ion at two pH values. The binding affinities of hydroxamate siderophores to UO2(2+) ions were observed to decrease with increasing Fe(III)Cl3 concentration at the lower pH. On the other hand, decreasing the pH has a smaller impact on the binding affinities between carboxylate siderophores and the UO2(2+) ion. Desmalonichrome in particular was shown to have the greatest relative affinity for UO2 at all pH and Fe(III) concentrations examined. These results suggest that acidic functional groups in the ligands are important for strong chelation with UO2 at lower pH. PMID:27232848

  15. Photoaffinity labelling of high affinity dopamine binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-03-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-)N'-4-azidobenzamidol)-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using (/sup 3/H)-SCH 23390 (a D/sub 1/ specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked (/sup 3/H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of (/sup 3/H)-SCH 23390 binding. Compounds which compete for D/sub 1/ receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D/sub 1/ (adenylate cyclase linked) dopamine receptor.

  16. Experimental and theoretical characterization of the high-affinity cation binding site of the purple membrane

    OpenAIRE

    Pardo, Leonardo; Sepulcre Sánchez, Francesc; Cladera Cerdà, Josep Bartomeu; Duñach, Mireia; Labarta, A.; Tejada, J.; Padrós Morell, Esteve

    1998-01-01

    Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff b...

  17. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

    Science.gov (United States)

    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

  18. Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter.

    Science.gov (United States)

    Samsudin, Firdaus; Parker, Joanne L; Sansom, Mark S P; Newstead, Simon; Fowler, Philip W

    2016-02-18

    Membrane transporters are critical modulators of drug pharmacokinetics, efficacy, and safety. One example is the proton-dependent oligopeptide transporter PepT1, also known as SLC15A1, which is responsible for the uptake of the ?-lactam antibiotics and various peptide-based prodrugs. In this study, we modeled the binding of various peptides to a bacterial homolog, PepTSt, and evaluated a range of computational methods for predicting the free energy of binding. Our results show that a hybrid approach (endpoint methods to classify peptides into good and poor binders and a theoretically exact method for refinement) is able to accurately predict affinities, which we validated using proteoliposome transport assays. Applying the method to a homology model of PepT1 suggests that the approach requires a high-quality structure to be accurate. Our study provides a blueprint for extending these computational methodologies to other pharmaceutically important transporter families. PMID:27028887

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

    OpenAIRE

    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...

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

    OpenAIRE

    Hong, Lian; Simon, John D.

    2007-01-01

    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 u...

  1. Isothermal Chemical Denaturation to Determine Binding Affinity of Small Molecules to G-Protein Coupled Receptors

    OpenAIRE

    Ross, Patrick; Weihofen, Wilhelm; Siu, Fai; Xie, Amy; Katakia, Hetal; Wright, S. Kirk; Hunt, Ian; Brown, Richard K; Freire, Ernesto

    2014-01-01

    The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation (ICD) has been shown to be a valuable biophysical method to determine in a direct and label-free fashion the binding of ligands to soluble proteins. In this communication, the application of isothermal che...

  2. Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

    Science.gov (United States)

    Ngo, That T; Narinesingh, Dyer

    2008-01-01

    The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels. PMID:18080884

  3. The next generation of transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Anthony Mathelier

    Full Text Available Finding where transcription factors (TFs bind to the DNA is of key importance to decipher gene regulation at a transcriptional level. Classically, computational prediction of TF binding sites (TFBSs is based on basic position weight matrices (PWMs which quantitatively score binding motifs based on the observed nucleotide patterns in a set of TFBSs for the corresponding TF. Such models make the strong assumption that each nucleotide participates independently in the corresponding DNA-protein interaction and do not account for flexible length motifs. We introduce transcription factor flexible models (TFFMs to represent TF binding properties. Based on hidden Markov models, TFFMs are flexible, and can model both position interdependence within TFBSs and variable length motifs within a single dedicated framework. The availability of thousands of experimentally validated DNA-TF interaction sequences from ChIP-seq allows for the generation of models that perform as well as PWMs for stereotypical TFs and can improve performance for TFs with flexible binding characteristics. We present a new graphical representation of the motifs that convey properties of position interdependence. TFFMs have been assessed on ChIP-seq data sets coming from the ENCODE project, revealing that they can perform better than both PWMs and the dinucleotide weight matrix extension in discriminating ChIP-seq from background sequences. Under the assumption that ChIP-seq signal values are correlated with the affinity of the TF-DNA binding, we find that TFFM scores correlate with ChIP-seq peak signals. Moreover, using available TF-DNA affinity measurements for the Max TF, we demonstrate that TFFMs constructed from ChIP-seq data correlate with published experimentally measured DNA-binding affinities. Finally, TFFMs allow for the straightforward computation of an integrated TF occupancy score across a sequence. These results demonstrate the capacity of TFFMs to accurately model DNA

  4. High-affinity dextromethorphan binding sites in guinea pig brain. II. Competition experiments.

    Science.gov (United States)

    Craviso, G L; Musacchio, J M

    1983-05-01

    Binding of dextromethorphan (DM) to guinea pig brain is stereoselective, since levomethorphan is 20 times weaker than DM in competing for DM sites. In general, opiate agonists and antagonists as well as their corresponding dextrorotatory isomers are weak competitors for tritiated dextromethorphan ([3H]DM) binding sites and display IC50 values in the micromolar range. In contrast, several non-narcotic, centrally acting antitussives are inhibitory in the nanomolar range (IC50 values for caramiphen, carbetapentane, dimethoxanate, and pipazethate are 25 nM, 9 nM, 41 nM, and 190 nM, respectively). Other antitussives, such as levopropoxyphene, chlophedianol, and fominoben, have poor affinity for DM sites whereas the antitussive noscapine enhances DM binding by increasing the affinity of DM for its central binding sites. Additional competition studies indicate that there is no correlation of DM binding with any of the known or putative neurotransmitters in the central nervous system. DM binding is also not related to tricyclic antidepressant binding sites or biogenic amine uptake sites. However, certain phenothiazine neuroleptics and typical and atypical antidepressants inhibit binding with IC50 values in the nanomolar range. Moreover, the anticonvulsant drug diphenylhydantoin enhances DM binding in a manner similar to that of noscapine. Preliminary experiments utilizing acid extracts of brain have not demonstrated the presence of an endogenous ligand for DM sites. The binding characteristics of DM sites studied in rat and mouse brain indicate that the relative potencies of several antitussives to inhibit specific DM binding vary according to species. High-affinity, saturable, and stereoselective [3H]DM binding sites are present in liver homogenates, but several differences have been found for these peripheral binding sites and those described for brain. Although the nature of central DM binding sites is not known, the potent interaction of several classes of centrally

  5. Binding energies and electron affinities of small silicon clusters (n=2--5)

    International Nuclear Information System (INIS)

    The Gaussian-2 (G2) theoretical procedure, based on ab initio molecular orbital theory, is used to calculate the energies of Sin and Si-n (n=1--5) clusters. The G2 energies are used to derive accurate binding energies and electron affinities of these clusters. The calculated electron affinities of Si2--Si4 are in agreement to within 0.1 eV with results from recent photoelectron spectroscopic measurements

  6. A high affinity binding site for cytokinin to a particulate fraction in carrot suspension cells

    International Nuclear Information System (INIS)

    Carrot suspension cells contain one class of high affinity binding sites for cytokinin in an 80,000 X g particulate fraction. Binding of [8-14C] - benzylaminopurine (BA) to this fraction assayed by a sedimentation method was found to be optimal at ph 6.0 and thermolabile. Specific binding was proved in competition experiments in which labelled BA was displaced by increasing concentrations of unlabelled BA. Scatchard plots of these results displayed a dissociation constant (Ksub(d)) of 33+- 6 n.M. The number of binding sites found was 1,100+-120 fmol g-1 fresh weight which is equivalent to a frequency of 23,000 binding sites per cell. The specificity of the binding sites to cytokinins and their analogues followed the sequence BA with highest affinity, kinetin, zeatin, iP and adenine. The cytokinin ribosides generally had a lower affinity than their cytokinin bases, and the affinity decreased in the order [9 R] BA, [9 R] iP, [i R]Z, [9 R] A. (author)

  7. Improved binding affinity and interesting selectivities of aminopyrimidine-bearing carbohydrate receptors in comparison with their aminopyridine analogues.

    Science.gov (United States)

    Lippe, Jan; Seichter, Wilhelm; Mazik, Monika

    2015-12-28

    Due to the problems with the exact prediction of the binding properties of an artificial carbohydrate receptor, the identification of characteristic structural features, having the ability to influence the binding properties in a predictable way, is of high importance. The purpose of our investigation was to examine whether the previously observed higher affinity of 2-aminopyrimidine-bearing carbohydrate receptors in comparison with aminopyridine substituted analogues represents a general tendency of aminopyrimidine-bearing compounds. Systematic binding studies on new compounds consisting of 2-aminopyrimidine groups confirmed such a tendency and allowed the identification of interesting structure-activity relationships. Receptors having different symmetries showed systematic preferences for specific glycosides, which are remarkable for such simple receptor systems. Particularly suitable receptor architectures for the recognition of selected glycosides were identified and represent a valuable base for further developments in this field. PMID:26467387

  8. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    Sumedha Roy; Shayoni Dutta; Kanika Khanna; Shruti Singla; Durai Sundar

    2012-07-01

    Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions −1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure–function relationships of the existing zinc finger–DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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 [125I]ovine PRL [(125I]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 125I using the chloramine-T method. Covalent labeling of PRL receptors with [125I]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 [125I]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 [125I]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

  11. Tension-compression asymmetry in the binding affinity of membrane-anchored receptors and ligands

    Science.gov (United States)

    Xu, Guang-Kui; Liu, Zishun; Feng, Xi-Qiao; Gao, Huajian

    2016-03-01

    Cell adhesion plays a crucial role in many biological processes of cells, e.g., immune responses, tissue morphogenesis, and stem cell differentiation. An essential problem in the molecular mechanism of cell adhesion is to characterize the binding affinity of membrane-anchored receptors and ligands under different physiological conditions. In this paper, a theoretical model is presented to study the binding affinity between a large number of anchored receptors and ligands under both tensile and compressive stresses, and corroborated by demonstrating excellent agreement with Monte Carlo simulations. It is shown that the binding affinity becomes lower as the magnitude of the applied stress increases, and drops to zero at a critical tensile or compressive stress. Interestingly, the critical compressive stress is found to be substantially smaller than the critical tensile stress for relatively long and flexible receptor-ligand complexes. This counterintuitive finding is explained by using the Euler instability theory of slender columns under compression. The tension-compression asymmetry in the binding affinity of anchored receptors and ligands depends subtly on the competition between the breaking and instability of their complexes. This study helps in understanding the role of mechanical forces in cell adhesion mediated by specific binding molecules.

  12. A CBM20 low-affinity starch-binding domain from glucan, water dikinase

    DEFF Research Database (Denmark)

    Christiansen, Camilla; Abou Hachem, Maher; Glaring, M.A.;

    2009-01-01

    from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent......The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that...... protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta....

  13. Engineering of binding affinity at metal ion binding sites for the stabilization of proteins: Subtilisin as a test case

    International Nuclear Information System (INIS)

    A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65/degrees/C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range of 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log K/sub a/ = 1.49. A series of refined X-ray crystal structures of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein. X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 /Angstrom/, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory. Moreover, when both Asp changes were introduced together, the binding affinity for Ca2+ was found to be increased about 6-fold over that for the wild-type protein, suggesting an independent and nearly additive effect on the total electrostatic potential at this locus

  14. Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues.

    Science.gov (United States)

    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. PMID:26525852

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

    International Nuclear Information System (INIS)

    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 1010 M-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 (r2>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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Ikegami Toru

    2001-09-01

    Full Text Available Abstract 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.

  20. Relative binding affinity of diethylstilbestrol to uterine nuclear estrogen receptor: effect of serum and serum albumin

    International Nuclear Information System (INIS)

    The relative binding affinity (RBA) of tritium labelled diethylstilbestrol (DES) was determined in nuclear fractions of rat uterus. DES displayed a two- to threefold greater affinity than estradiol for nuclear E receptor. The RBA of DES to nuclear E receptor was lowered significantly in the presence of rat serum (43 +/- 1) or human serum (52 +/- 7). Dilution of human serum resulted in a progressive increase in the RBA of DES which approached that observed in the absence of serum. Addition of purified human serum albumin mimicked the decrease in RBA of DES that was observed with serum. The IC50 of estradiol was not changed in the presence of either rat serum or albumin. These data show that DES possesses a greater affinity for nuclear E receptor than estradiol and that serum albumin can modulate DES binding to uterine E receptor

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

    International Nuclear Information System (INIS)

    Binding of [3H]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 [3H]cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 950C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of [3H]cocaine (15 nM) was inhibited by increasing concentrations of Na+ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific [3H]cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC50 = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting [3H]cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC50 values below μM concentrations

  2. Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: selective loss of agonist low-affinity and pirenzepine high-affinity binding sites

    International Nuclear Information System (INIS)

    The effects of brief incubation with carbamylcholine on subsequent binding of [3H]N-methylscopolamine were investigated in mouse neuroblastoma cells (clone N1E-115). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both pirenzepine and carbamylcholine interacted with high- and low-affinity subsets of muscarinic receptors. Computer-assisted analysis of the competition between pirenzepine and carbamylcholine with [3H]N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various muscarinic receptor agonists to induce a decrease in consequent [3H]N-methylscopolamine binding and their efficacy in stimulating cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in cyclic GMP levels were capable of diminishing ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of muscarinic receptor-mediated cyclic GMP formation

  3. Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

    OpenAIRE

    Nakagama, H; Heinrich, G.; Pelletier, J; Housman, D E

    1995-01-01

    The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, settin...

  4. Is There Consistency between the Binding Affinity and Inhibitory Potential of Natural Polyphenols as α-amylase Inhibitors?

    Science.gov (United States)

    Xu, Wei; Shao, Rong; Xiao, Jianbo

    2016-07-26

    The inhibitory potential of natural polyphenols for α-amylases has attracted great interests among researchers. The structure-affinity properties of natural polyphenols binding to α-amylase and the structure-activity relationship of dietary polyphenols inhibiting α-amylase were deeply investigated. There is a lack of consistency between the structure-affinity relationship and the structure-activity relationship of natural polyphenols as α-amylase inhibitors. Is it consistent between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors? It was found that the consistency between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors is not equivocal. For example, there is no consistency between the binding affinity and the inhibitory potential of quercetin and its glycosides as α-amylase inhibitors. However, catechins with higher α-amylase inhibitory potential exhibited higher affinity with α-amylase. PMID:25748632

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

    International Nuclear Information System (INIS)

    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, [125I]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 [125I]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

  6. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    XIANG YongZhe; WANG Na; ZHANG Ji; LI Kun; ZHANG ZhongWei; LIN HongHui; YU XiaoQi

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction be-tween 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane.High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA con-densation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

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

    Science.gov (United States)

    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

  8. Ligand Binding Affinities of Arctigenin and Its Demethylated Metabolites to Estrogen Receptor Alpha

    Directory of Open Access Journals (Sweden)

    Masao Hattori

    2013-01-01

    Full Text Available 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 IC50 value of (2R,3R-2-(4-hydroxy-3-methoxybenzyl-3-(3,4-dihydroxybenzyl-butyrolactone was 7.9 × 10−4 M.

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

    International Nuclear Information System (INIS)

    The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBDKZ), originating from Pseudomonas aeruginosa bacteriophage φKZ, has been examined using a fusion protein of PBDKZ and green fluorescent protein (PBDKZ-GFP). A fluorescence recovery after photobleaching analysis of bound PBDKZ-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 107 M-1 for the PBDKZ-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 (PBDKZ-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.

  10. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kuziemko, G.M.; Stroh, M.; Stevens, R.C. [Univ. of California, Berkeley, CA (United States)]|[Lawrence Berkeley National Lab., CA (United States)

    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.

  12. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

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

    Science.gov (United States)

    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.

  14. A community resource benchmarking predictions of peptide binding to MHC-I molecules.

    OpenAIRE

    Bjoern Peters; Huynh-Hoa Bui; Sune Frankild; Morten Nielson; Claus Lundegaard; Emrah Kostem; Derek Basch; Kasper Lamberth; Mikkel Harndahl; Ward Fleri; Wilson, Stephen S; John Sidney; Ole Lund; Soren Buus; Alessandro Sette

    2006-01-01

    Recognition of peptides bound to major histocompatibility complex (MHC) class I molecules by T lymphocytes is an essential part of immune surveillance. Each MHC allele has a characteristic peptide binding preference, which can be captured in prediction algorithms, allowing for the rapid scan of entire pathogen proteomes for peptide likely to bind MHC. Here we make public a large set of 48,828 quantitative peptide-binding affinity measurements relating to 48 different mouse, human, macaque, an...

  15. A community resource benchmarking predictions of peptide binding to MHC-I molecules

    OpenAIRE

    Peters, B; Bui, HH; Pletscher-Frankild, Sune; Nielsen, Morten; Lundegaard, Claus; Kostem, E; Basch, D; Lamberth, K.; Harndahl, M.; Fleri, W.; Wilson, SS; Sidney, J; Lund, Ole; Buus, S.; Sette, Alessandro

    2006-01-01

    Recognition of peptides bound to major histocompatibility complex (MHC) class I molecules by T lymphocytes is an essential part of immune surveillance. Each MHC allele has a characteristic peptide binding preference, which can be captured in prediction algorithms, allowing for the rapid scan of entire pathogen proteomes for peptide likely to bind MHC. Here we make public a large set of 48,828 quantitative peptide-binding affinity measurements relating to 48 different mouse, human, macaque, an...

  16. A Community Resource Benchmarking Predictions of Peptide Binding to MHC-I Molecules

    OpenAIRE

    Bjoern Peters; Huynh-Hoa Bui; Sune Frankild; Morten Nielson; Claus Lundegaard; Emrah Kostem; Derek Basch; Kasper Lamberth; Mikkel Harndahl; Ward Fleri; Wilson, Stephen S; John Sidney; Ole Lund; Soren Buus; Alessandro Sette

    2006-01-01

    Recognition of peptides bound to major histocompatibility complex (MHC) class I molecules by T lymphocytes is an essential part of immune surveillance. Each MHC allele has a characteristic peptide binding preference, which can be captured in prediction algorithms, allowing for the rapid scan of entire pathogen proteomes for peptide likely to bind MHC. Here we make public a large set of 48,828 quantitative peptide-binding affinity measurements relating to 48 different mouse, human, macaque, an...

  17. An in silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-peptide interactions.

    Directory of Open Access Journals (Sweden)

    Garima Tiwari

    Full Text Available Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K(i or K(d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K(i or K(d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors

  18. Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

    OpenAIRE

    Williams, R K; Straus, S E

    1997-01-01

    Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the s...

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

    OpenAIRE

    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 Ce...

  20. Point mutations of human interleukin-1 with decreased receptor binding affinity

    International Nuclear Information System (INIS)

    Interleukin-1 (IL-1) is a monocyte-derived polypeptide hormone that interacts with a plasma membrane receptor. We have used oligonucleotide-directed mutagenesis to construct mutant human IL-1 proteins. Three different point mutants in a unique histidine residue (position 30) exhibited varying degrees of reduced IL-1 receptor binding affinity, whereas point mutants at five other residues behaved normally. Structural analysis of these mutant proteins by nuclear magnetic resonance spectroscopy detected no (or only minor) conformational changes relative to wild-type IL-1. These data suggest that the unique histidine residue influences the architecture of the receptor binding site on human IL-1. (Auth.)

  1. Assessment of Binding Affinity between Drugs and Human Serum Albumin Using Nanoporous Anodic Alumina Photonic Crystals.

    Science.gov (United States)

    Nemati, Mahdieh; Santos, Abel; Law, Cheryl Suwen; Losic, Dusan

    2016-06-01

    In this study, we report an innovative approach aiming to assess the binding affinity between drug molecules and human serum albumin by combining nanoporous anodic alumina rugate filters (NAA-RFs) modified with human serum albumin (HSA) and reflectometric interference spectroscopy (RIfS). NAA-RFs are photonic crystal structures produced by sinusoidal pulse anodization of aluminum that present two characteristic optical parameters, the characteristic reflection peak (λPeak), and the effective optical thickness of the film (OTeff), which can be readily used as sensing parameters. A design of experiments strategy and an ANOVA analysis are used to establish the effect of the anodization parameters (i.e., anodization period and anodization offset) on the sensitivity of HSA-modified NAA-RFs toward indomethacin, a model drug. To this end, two sensing parameters are used, that is, shifts in the characteristic reflection peak (ΔλPeak) and changes in the effective optical thickness of the film (ΔOTeff). Subsequently, optimized NAA-RFs are used as sensing platforms to determine the binding affinity between a set of drugs (i.e., indomethacin, coumarin, sulfadymethoxine, warfarin, and salicylic acid) and HSA molecules. Our results verify that the combination of HSA-modified NAA-RFs with RIfS can be used as a portable, low-cost, and simple system for establishing the binding affinity between drugs and plasma proteins, which is a critical factor to develop efficient medicines for treating a broad range of diseases and medical conditions. PMID:27128744

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

    Science.gov (United States)

    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

  3. Engineering protein therapeutics: predictive performances of a structure-based virtual affinity maturation protocol.

    Science.gov (United States)

    Oberlin, Michael; Kroemer, Romano; Mikol, Vincent; Minoux, Hervé; Tastan, Erdogan; Baurin, Nicolas

    2012-08-27

    The implementation of a structure based virtual affinity maturation protocol and evaluation of its predictivity are presented. The in silico protocol is based on conformational sampling of the interface residues (using the Dead End Elimination/A* algorithm), followed by the estimation of the change of free energy of binding due to a point mutation, applying MM/PBSA calculations. Several implementations of the protocol have been evaluated for 173 mutations in 7 different protein complexes for which experimental data were available: the use of the Boltzamnn averaged predictor based on the free energy of binding (ΔΔG(*)) combined with the one based on its polar component only (ΔΔE(pol*)) led to the proposal of a subset of mutations out of which 45% would have successfully enhanced the binding. When focusing on those mutations that are less likely to be introduced by natural in vivo maturation methods (99 mutations with at least two base changes in the codon), the success rate is increased to 63%. In another evaluation, focusing on 56 alanine scanning mutations, the in silico protocol was able to detect 89% of the hot-spots. PMID:22788756

  4. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  5. Contribution of a p75 interleukin 2 binding peptide to a high-affinity interleukin 2 receptor complex

    International Nuclear Information System (INIS)

    There are at least two forms of cellular receptors for interleukin 2(IL-2); one with a very high affinity and the other with a lower affinity. The authors identified a non-Tac IL-2 binding peptide with a relative molecular weight of 75,000 (p75). Cell lines bearing either the p55 Tac or the p75 peptide alone manifested low-affinity IL-2 binding, whereas a cell line bearing both peptides manifested both high- and low-affinity receptors. Cross-linking studies were performed with 125I-labeled IL-2. After the internalization of labeled IL-2 through high-affinity receptors, the p75 peptide could not be detected by cross-linking studies. Furthermore, fusion of cell membranes from low-affinity IL-2 binding cell lines bearing the Tac peptide alone with membranes from a cell line bearing the p75 peptide alone generated hybrid membranes bearing high-affinity receptors. These results suggest a multichain model for the high-affinity Il-2 receptor in which high-affinity receptors would be expressed when both Tac and p75 Il-2 binding peptides are present and associated in a receptor complex

  6. Characterization of high affinity [3H]triazolam binding in rat brain

    International Nuclear Information System (INIS)

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on [3H]TZ binding. Saturation studies showed a shift to lower affinity at 370C (K/sub d/ = 0.25 +/- 0.01 nM at O0C; K/sub d/ = 1.46 +/- 0.03 nM at 370C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 00C and 1001 +/- 43 fmoles/mg prot. at 370C). Inhibition studies showed that [3H]TZ binding displayed no GABA shift at 00C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 370C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 370C. In Ro 15-1788/[3H]TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on [3H]TZ binding at both temperatures. In conclusion [3H]TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists

  7. Sensitivity of binding of high-affinity dopamine receptor radioligands to increased synaptic dopamine.

    Science.gov (United States)

    Gatley, S J; Gifford, A N; Carroll, F I; Volkow, N D

    2000-12-15

    PET and SPECT studies have documented that D2 radioligands of moderate affinity, but not radioligands of high affinity, are sensitive to pharmacological challenges that alter synaptic dopamine levels. The objective of this work was to determine whether the brain kinetics of high-affinity radioligands for dopamine D1 ([(3)H]SCH 23390) and D2 ([(123)I]epidepride) receptors were altered by a prolonged elevation of synaptic dopamine induced by the potent cocaine analog RTI-55. Mice were injected intravenously with radioligands either 30 min after or 4 h before intraperitoneal administration of RTI-55 (2 mg/kg). In separate experiments, the pharmacological effects of RTI-55 were assessed biochemically by measuring uptake of dopamine in synaptosomes prepared from RTI-treated mice and behaviorally by monitoring locomotor activity. Consistent with the expected elevation of synaptic dopamine, RTI-55 induced a long-lasting decrement in dopamine uptake measured ex vivo, and a prolonged increase in locomotor activity. RTI-55 injected prior to the radioligands induced a significant (P epidepride at 15 min, relative to saline-treated controls, but there were no differences between the two groups at later time-points. For [(3)H]SCH 23390, both initial striatal uptake and subsequent clearance were slightly increased by preadministration of RTI-55. Administration of RTI-55 4 h after the radioligands (i.e., when it was presumed that a state of near equilibrium binding of the radioligands had been reached), was associated with a significant reduction of striatal radioactivity for both radiotracers. Our results are consistent with increased competition between dopamine and radioligand for binding to both D1 and D2 receptors after treatment with RTI-55. We suggest that the magnitude of the competition is reduced by failure of the receptor binding of high-affinity radioligands to rapidly attain equilibrium. PMID:11044896

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

    International Nuclear Information System (INIS)

    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, 3H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a 3H-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 3H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A4, 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

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

    International Nuclear Information System (INIS)

    cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with 32P-cGMP and quantified by retention of complexes on nitrocellulose filters. By PAL, high affinity sites were present on the α and β 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 γ/ photocrosslinked with PDE/sub α/ or PDE/sub β/, respectively. cGMP prebound to these high affinity sites was released by light-activated G-protein or its α subunit complexed with GTPγS; 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

  10. Compressed images for affinity prediction-2 (CIFAP-2): an improved machine learning methodology on protein-ligand interactions based on a study on caspase 3 inhibitors.

    Science.gov (United States)

    Erdas, Ozlem; Andac, Cenk A; Gurkan-Alp, A Selen; Alpaslan, Ferda Nur; Buyukbingol, Erdem

    2015-01-01

    The aim of this study is to propose an improved computational methodology, which is called Compressed Images for Affinity Prediction-2 (CIFAP-2) to predict binding affinities of structurally related protein-ligand complexes. CIFAP-2 method is established based on a protein-ligand model from which computational affinity information is obtained by utilizing 2D electrostatic potential images determined for the binding site of protein-ligand complexes. The quality of the prediction of the CIFAP-2 algorithm was tested using partial least squares regression (PLSR) as well as support vector regression (SVR) and adaptive neuro-fuzzy ınference system (ANFIS), which are highly promising prediction methods in drug design. CIFAP-2 was applied on a protein-ligand complex system involving Caspase 3 (CASP3) and its 35 inhibitors possessing a common isatin sulfonamide pharmacophore. As a result, PLSR affinity prediction for the CASP3-ligand complexes gave rise to the most consistent information with reported empirical binding affinities (pIC(50)) of the CASP3 inhibitors. PMID:25578823

  11. Tuning affinity and reversibility for O2 binding in dinuclear Co(II) complexes

    DEFF Research Database (Denmark)

    Vad, Mads Sørensen; Johansson, Frank Bartnik; Seidler-Egdal, Rune Kirk;

    2013-01-01

    The O2 binding affinity of a series of dicobalt(II) complexes can be tuned between p(O2)50% = 2.3 × 10−3 and 700 × 10−3 atm at 40 °C by varying the number of H and Cl atoms in the bridging acetato ligands of [Co2(bpbp)(CH(3−n)ClnCO2)(CH3CN)2]2+, where bpbp− = 2,6-bis(N,N-bis(2-pyridylmethyl......)aminomethyl)-4- tert-butylphenolate and n = {0, 1, 2, 3}. O2 binds most strongly to the deoxy complex containing the acetato bridge and the O2 affinity decreases linearly as the number of Cl atoms is increased from 0 to 3 in [Co2(bpbp)(O2)(CH3CO2)]2+, [Co2(bpbp)(O2)(CH2ClCO2)]2+, [Co2(bpbp)(O2)(CHCl2CO2)]2+ and...... acetato-bridged complex to 696 mV for the trichloroacetato-bridged system. Despite the clear difference in reactivity in solution, there are no clear trends which can be correlated to O2 affinity in the O–O bond lengths in the X-ray crystal structures at 180 K (1.415(4)–1.424(2) Å) or in the frequencies...

  12. New beginnings for matrix metalloproteinase inhibitors: identification of high-affinity zinc-binding groups.

    Science.gov (United States)

    Puerta, David T; Lewis, Jana A; Cohen, Seth M

    2004-07-14

    In an effort to identify promising non-hydroxamate inhibitors of matrix metalloproteinases (MMPs), several new zinc-binding groups (ZBGs) based on pyrone, pyrothione, hydroxypyridinone, and hydroxypyridinethione chelators have been examined. Structural studies with tris(pyrazolyl)borate model complexes show that these ligands bind to the MMP active site zinc(II) ion in a bidentate fashion, similar to that found with hydroxamate-based inhibitors. Fluorescence- and colorimetric-based enzyme assays have been used to determine the IC50 values for these ZBGs against MMP-3; mixed O,S-donor ligands were found to be remarkably potent, with IC50 values as much as 700-fold lower than that found for acetohydroxamic acid. Inhibitory activity was found to parallel metal binding affinity as determined in titrations with model complexes. These results demonstrate that MPIs based on new ZBGs are feasible and may indeed improve the overall performance of inhibitors designed against these important medicinal targets. PMID:15237990

  13. Prediction of MHC binding peptides and epitopes from alfalfa mosaic virus.

    Science.gov (United States)

    Gomase, Virendra S; Kale, Karbhari V; Chikhale, Nandkishor J; Changbhale, Smruti S

    2007-08-01

    Peptide fragments from alfalfa mosaic virus involved multiple antigenic components directing and empowering the immune system to protect the host from infection. MHC molecules are cell surface proteins, which take active part in host immune reactions and involvement of MHC class-I & II in response to almost all antigens. Coat protein of alfalfa mosaic virus contains 221 aa residues. Analysis found five MHC ligands in coat protein as 64-LSSFNGLGV-72; 86- RILEEDLIY-94; 96-MVFSITPSY-104; 100- ITPSYAGTF-108; 110- LTDDVTTED-118; having rescaled binding affinity and c-terminal cleavage affinity more than 0.5. The predicted binding affinity is normalized by the 1% fractil. The MHC peptide binding is predicted using neural networks trained on c-terminals of known epitopes. In analysis predicted MHC/peptide binding is a log transformed value related to the IC50 values in nM units. Total numbers of peptides found are 213. Predicted MHC binding regions act like red flags for antigen specific and generate immune response against the parent antigen. So a small fragment of antigen can induce immune response against whole antigen. This theme is implemented in designing subunit and synthetic peptide vaccines. The sequence analysis method allows potential drug targets to identify active sites against plant diseases. The method integrates prediction of peptide MHC class I binding; proteosomal c-terminal cleavage and TAP transport efficiency. PMID:17691913

  14. Identification of pyrazosulfuron-ethyl binding affinity and binding site subdomain IIA in human serum albumin by spectroscopic methods

    Science.gov (United States)

    Ding, Fei; Liu, Wei; Zhang, Xi; Wu, Li-Jun; Zhang, Li; Sun, Ying

    2010-03-01

    Pyrazosulfuron-ethyl (PY) is a sulfonylurea herbicide developed by DuPont which has been widely used for weed control in cereals. The determination of PY binding affinity and binding site in human serum albumin (HSA) by spectroscopic methods is the subject of this work. From the fluorescence emission, circular dichroism and three-dimensional fluorescence results, the interaction of PY with HSA caused secondary structure changes in the protein. Fluorescence data demonstrated that the quenching of HSA fluorescence by PY was the result of the formation of HSA-PY complex at 1:1 molar ratio, a static mechanism was confirmed to lead to the fluorescence quenching. Hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement results show that hydrophobic patches are the major sites for PY binding on HSA. The thermodynamic parameters Δ H° and Δ S° were calculated to be -36.32 kJ mol -1 and -35.91 J mol -1 K -1, which illustrated van der Waals forces and hydrogen bonds interactions were the dominant intermolecular force in stabilizing the complex. Also, site marker competitive experiments showed that the binding of PY to HSA took place primarily in subdomain IIA (Sudlow's site I). What presented in this paper binding research enriches our knowledge of the interaction between sulfonylurea herbicides and the physiologically important protein HSA.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    International Nuclear Information System (INIS)

    The mechanism by which delta9 tetrahydrocannabinol (delta9THC), 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'-Trimethylammonium-delta8THC (TMA) is a positively charged analog of delta-8THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [3H]-5'-trimethylammonium-delta-8THC ([3H]TMA) to rat neuronal membranes. [3H]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 [3H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

  17. Influence of target concentration and background binding on in vitro selection of affinity reagents.

    Directory of Open Access Journals (Sweden)

    Jinpeng Wang

    Full Text Available Nucleic acid-based aptamers possess many useful features that make them a promising alternative to antibodies and other affinity reagents, including well-established chemical synthesis, reversible folding, thermal stability and low cost. However, the selection process typically used to generate aptamers (SELEX often requires significant resources and can fail to yield aptamers with sufficient affinity and specificity. A number of seminal theoretical models and numerical simulations have been reported in the literature offering insights into experimental factors that govern the effectiveness of the selection process. Though useful, these previous models have not considered the full spectrum of experimental factors or the potential impact of tuning these parameters at each round over the course of a multi-round selection process. We have developed an improved mathematical model to address this important question, and report that both target concentration and the degree of non-specific background binding are critical determinants of SELEX efficiency. Although smaller target concentrations should theoretically offer superior selection outcome, we show that the level of background binding dramatically affect the target concentration that will yield maximum enrichment at each round of selection. Thus, our model enables experimentalists to determine appropriate target concentrations as a means for protocol optimization. Finally, we perform a comparative analysis of two different selection methods over multiple rounds of selection, and show that methods with inherently lower background binding offer dramatic advantages in selection efficiency.

  18. A new BODIPY/nanoparticle/Ni affinity system for binding of cytochrome c

    International Nuclear Information System (INIS)

    Highlights: • BODIPY was synthesized, and then attached to magnetic nanoparticles. • Ni(II) ions were chelated on prepared material. • The binding of cytochrome c to obtained material was studied. - Abstract: In this study, 3,5-{Bis[4,4-difluoro, 8-(2,6-diethyl, 1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene)]}benzoylchloride (BODIPY) was synthesized for the improving of a new immobilized metal affinity supporting material. Firstly, the synthesized BODIPY was immobilized on iron oxide superparamagnetic nanoparticles (SPIONs) and then, Ni(II) ions were chelated with the active terminals of BODIPY on nanoparticles surfaces to prepare an immobilized metal affinity (IMA) adsorbent for protein adsorption. The amount of BODIPY coated on SPIONs was about 29.7 μM at 10 mg nanoparticles. 738 μmol of Ni(II) ions were loaded to 10 mg of the SPIONs/BODIPY. The binding amount of cytochrome c was found to be 170 μg to the SPIONs/BODIPY/Ni at pH 7.4. The binding amount of the molecules on SPIONs was analyzed by using UV–vis, fluorescence and atomic absorption spectroscopy. The characterization of the prepared surfaces was performed by FT-IR, SEM and TEM

  19. A new BODIPY/nanoparticle/Ni affinity system for binding of cytochrome c

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Kursunlu, Ahmed Nuri [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Arslan, Gulsin [Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey)

    2015-09-15

    Highlights: • BODIPY was synthesized, and then attached to magnetic nanoparticles. • Ni(II) ions were chelated on prepared material. • The binding of cytochrome c to obtained material was studied. - Abstract: In this study, 3,5-{Bis[4,4-difluoro, 8-(2,6-diethyl, 1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene)]}benzoylchloride (BODIPY) was synthesized for the improving of a new immobilized metal affinity supporting material. Firstly, the synthesized BODIPY was immobilized on iron oxide superparamagnetic nanoparticles (SPIONs) and then, Ni(II) ions were chelated with the active terminals of BODIPY on nanoparticles surfaces to prepare an immobilized metal affinity (IMA) adsorbent for protein adsorption. The amount of BODIPY coated on SPIONs was about 29.7 μM at 10 mg nanoparticles. 738 μmol of Ni(II) ions were loaded to 10 mg of the SPIONs/BODIPY. The binding amount of cytochrome c was found to be 170 μg to the SPIONs/BODIPY/Ni at pH 7.4. The binding amount of the molecules on SPIONs was analyzed by using UV–vis, fluorescence and atomic absorption spectroscopy. The characterization of the prepared surfaces was performed by FT-IR, SEM and TEM.

  20. Ring size in cyclic endomorphin-2 analogs modulates receptor binding affinity and selectivity.

    Science.gov (United States)

    Piekielna, Justyna; Kluczyk, Alicja; Gentilucci, Luca; Cerlesi, Maria Camilla; Calo', Girolamo; Tomböly, Csaba; Łapiński, Krzysztof; Janecki, Tomasz; Janecka, Anna

    2015-06-01

    The study reports the solid-phase synthesis and biological evaluation of a series of new side chain-to-side chain cyclized opioid peptide analogs of the general structure Tyr-[D-Xaa-Phe-Phe-Asp]NH2, where Xaa = Lys (1), Orn (2), Dab (3), or Dap (4) (Dab = 2,4-diaminobutyric acid, Dap = 2,3-diaminopropionic acid), containing 17- to 14-membered rings. The influence of the ring size on binding to the MOP, DOP and KOP opioid receptors was studied. In general, the reduction of the size of the macrocyclic ring increased the selectivity for the MOP receptor. The cyclopeptide incorporating Xaa = Lys displayed subnanomolar MOP affinity but modest selectivity over the KOP receptor, while the analog with the Orn residue showed increased affinity and selectivity for MOP. The analog with Dab was a weak MOP agonist and did not bind to the other two opioid receptors. Finally, the peptide with Xaa = Dap was completely MOP receptor-selective with subnanomolar affinity. Interestingly, the deletion of one Phe residue from 1 led to the 14-membered Tyr-c[D-Lys-Phe-Asp]NH2 (5), a potent and selective MOP receptor ligand. The in vitro potencies of the new analogs were determined in a calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. A good correlation between binding and the functional test results was observed. The influence of the ring size, solid support and the N-terminal protecting group on the formation of cyclodimers was studied. PMID:25948019

  1. Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

    Science.gov (United States)

    Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

    2016-03-14

    The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds. PMID:26795018

  2. Affinity capillary electrophoresis applied to investigation of noncovalent bindings of (bio)molecules

    Czech Academy of Sciences Publication Activity Database

    Kašička, Václav; Norková, Renáta; Růžička, Martin; Koval, Dušan; Čížková, Martina; Teplý, Filip

    Pardubice: Univerzita Pardubice, 2014 - (Horna, A.; Jandera, P.). s. 39 ISBN 978-80-7395-777-3. [ISSS 2014. International Symposium on Separation Sciences /20./. 30.08.2014-02.09.2014, Praha] R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR(CZ) GA13-17224S; GA ČR GA13-32974S Institutional support: RVO:61388963 Keywords : affinity capillary electrophoresis * noncovalent interactions * binding constants Subject RIV: CB - Analytical Chemistry, Separation

  3. Theoretical prediction of the binding free energy for mutants of replication protein A.

    Science.gov (United States)

    Carra, Claudio; Saha, Janapriya; Cucinotta, Francis A

    2012-07-01

    The replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), single stranded DNA (ssDNA) binding protein required for pivotal functions in the cell metabolism, such as chromosomal replication, prevention of hairpin formation, DNA repair and recombination, and signaling after DNA damage. Studies based on deletions and mutations have identified the high affinity ssDNA binding domains in the 70 kDa subunit of RPA, regions A and B. Individually, the domain A and B have a low affinity for ssDNA, while tandems composed of AA, AB, BB, and BA sequences bind the ssDNA with moderate to high affinity. Single and double point mutations on polar residues in the binding domains leads to a reduction in affinity of RPA for ssDNA, in particular when two hydrophilic residues are involved. In view of these results, we performed a study based on molecular dynamics simulation aimed to reproduce the experimental change in binding free energy, ΔΔG, of RPA70 mutants to further elucidate the nature of the protein-ssDNA interaction. The MM-PB(GB)SA methods implemented in Amber10 and the code FoldX were used to estimate the binding free energy. The theoretical and experimental ΔΔG values correlate better when the results are obtained by MM-PBSA calculated on individual trajectories for each mutant. In these conditions, the correlation coefficient between experimental and theoretical ΔΔG reaches a value of 0.95 despite the overestimation of the energy change by one order of magnitude. The decomposition of the MM-GBSA energy per residue allows us to correlate the change of the affinity with the residue polarity and energy contribution to the binding. The method revealed reliable predictions of the change in the affinity in function of mutations, and can be used to identify new mutants with distinct binding properties. PMID:22160652

  4. In Silico and in Vitro Study of Binding Affinity of Tripeptides to Amyloid β Fibrils: Implications for Alzheimer's Disease.

    Science.gov (United States)

    Viet, Man Hoang; Siposova, Katarina; Bednarikova, Zuzana; Antosova, Andrea; Nguyen, Truc Trang; Gazova, Zuzana; Li, Mai Suan

    2015-04-23

    Self-assembly of Aβ peptides into amyloid aggregates has been suggested as the major cause of Alzheimer's disease (AD). Nowadays, there is no medication for AD, but experimental data indicate that reversion of the process of amyloid aggregation reduces the symptoms of disease. In this paper, all 8000 tripeptides were studied for their ability to destroy Aβ fibrils. The docking method and the more sophisticated MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method were employed to calculate the binding affinity and mode of tripeptides to Aβ fibrils. The ability of these peptides to depolymerize Aβ fibrils was also investigated experimentally using atomic force microscopy and fluorescence spectroscopy (Thioflavin T assay). It was shown that tripeptides prefer to bind to hydrophobic regions of 6Aβ9-40 fibrils. Tripeptides WWW, WWP, WPW and PWW were found to be the most potent binders. In vitro experiments showed that tight-binding tripeptides have significant depolymerizing activities and their DC50 values determined from dose-response curves were in micromolar range. The ability of nonbinding (GAM, AAM) and weak-binding (IVL and VLA) tripeptides to destroy Aβ fibrils was negligible. In vitro data of tripeptide depolymerizing activities support the predictions obtained by molecular docking and all-atom simulation methods. Our results suggest that presence of multiple complexes of heterocycles forming by tryptophan and proline residues in tripeptides is crucial for their tight binding to Aβ fibrils as well as for extensive fibril depolymerization. We recommend PWW for further studies as it has the lowest experimental binding constant. PMID:25815792

  5. An HIV-1 encoded peptide mimics the DNA binding loop of NF-κB and binds thioredoxin with high affinity

    International Nuclear Information System (INIS)

    Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-κB), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-κB via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-κB/Rel family of transcription factors by the putative HIV-1 pro-fs protein

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

    Directory of Open Access Journals (Sweden)

    Alizadeh Zahra

    2003-06-01

    Full Text Available Abstract 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.

  7. Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

    Directory of Open Access Journals (Sweden)

    Claudia Forero

    2000-06-01

    Full Text Available The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.

  8. Predicting binding free energies in solution

    CERN Document Server

    Jensen, Jan H

    2015-01-01

    Recent predictions of absolute binding free energies of host-guest complexes in aqueous solution using electronic structure theory have been encouraging for some systems, while other systems remain problematic for others. In paper I summarize some of the many factors that could easily contribute 1-3 kcal/mol errors at 298 K: three-body dispersion effects, molecular symmetry, anharmonicity, spurious imaginary frequencies, insufficient conformational sampling, wrong or changing ionization states, errors in the solvation free energy of ions, and explicit solvent (and ion) effects that are not well-represented by continuum models. While the paper is primarily a synthesis of previously published work there are two new results: the adaptation of Legendre transformed free energies to electronic structure theory and a use of water clusters that maximizes error cancellation in binding free energies computed using explicit solvent molecules. While I focus on binding free energies in aqueous solution the approach also a...

  9. Sourcing the affinity of flavonoids for the glycogen phosphorylase inhibitor site via crystallography, kinetics and QM/MM-PBSA binding studies: comparison of chrysin and flavopiridol.

    Science.gov (United States)

    Tsitsanou, Katerina E; Hayes, Joseph M; Keramioti, Maria; Mamais, Michalis; Oikonomakos, Nikos G; Kato, Atsushi; Leonidas, Demetres D; Zographos, Spyros E

    2013-11-01

    Flavonoids have been discovered as novel inhibitors of glycogen phosphorylase (GP), a target to control hyperglycemia in type 2 diabetes. To elucidate the mechanism of inhibition, we have determined the crystal structure of the GPb-chrysin complex at 1.9 Å resolution. Chrysin is accommodated at the inhibitor site intercalating between the aromatic side chains of Phe285 and Tyr613 through π-stacking interactions. Chrysin binds to GPb approximately 15 times weaker (Ki=19.01 μM) than flavopiridol (Ki=1.24 μM), exclusively at the inhibitor site, and both inhibitors display similar behavior with respect to AMP. To identify the source of flavopiridols' stronger affinity, molecular docking with Glide and postdocking binding free energy calculations using QM/MM-PBSA have been performed and compared. Whereas docking failed to correctly rank inhibitor binding conformations, the QM/MM-PBSA method employing M06-2X/6-31+G to model the π-stacking interactions correctly reproduced the experimental results. Flavopiridols' greater binding affinity is sourced to favorable interactions of the cationic 4-hydroxypiperidin-1-yl substituent with GPb, with desolvation effects limited by the substituent conformation adopted in the crystallographic complex. Further successful predictions using QM/MM-PBSA for the flavonoid quercetagetin (which binds at the allosteric site) leads us to propose the methodology as a useful and inexpensive tool to predict flavonoid binding. PMID:23279842

  10. Temperature-sensitive high affinity [3H]serotonin binding: characterization and effects of antidepressant treatment

    International Nuclear Information System (INIS)

    Characterization of temperature-sensitive [3H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S1 and S2 receptors. In vivo pretreatment (48 h before) with mianserin did not alter B/sub max/ or Kd for the 1 nM Kd [3H]5-HT site, although [3H]ketanserin (S2) densities were decreased by 50%. This suggested that possible S2 components of [3H]5-HT binding must be negligible, even though ketanserin competed with high affinity (IC50 = 3 nM) for a portion of the 1 nM Kd [3H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [3H]5-HT site in a non-competitive manner, as shown by a decrease in B/sub max/ with no change in Kd after in vitro incubation. The complex inhibition data may therefore represent indirect interactions through another site

  11. Application of a constrained regularization method to extraction of affinity distributions: proton and metal binding to humic substances.

    Science.gov (United States)

    Orsetti, Silvia; Andrade, Estela María; Molina, Fernando V

    2009-08-15

    The binding of proton and metal cations to humic substances has been analyzed with a regularized fitting procedure (using the CONTIN software package) to extract conditional affinity distributions, valid at a given ionic strength, from binding (titration) curves. The procedure was previously tested with simulated titration curves using a simple bi-Gaussian model, the NICA-Donnan model, and the Stockholm humic model. Application to literature data for proton binding shows that in several cases the affinity distribution found is bimodal (carboxylic and phenolic sites) as usually assumed; however in other cases, specially for fulvic acids, a trimodal distribution is clearly discerned, with a smaller peak between the two noted above attributed to the presence of vicinal carboxylic groups. The analysis of metal binding curves has been performed in a few cases where the available data could be reliably processed, separating the proton affinity distribution and obtaining the conditional affinity spectra. For Cd(II) and Pb(II) a bimodal distribution is found, attributed in principle to mono- and bidentate binding, based on spectroscopic data. In the case of Cu(II), a more complex affinity distribution is found showing 3-4 peaks; this is consistent with spectroscopic studies, where different binding modes, up to tetradentate, have been observed. PMID:19477457

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

    Science.gov (United States)

    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…

  13. Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays.

    Science.gov (United States)

    Crane, Jonathan M; Lam, Chiwah; Rossi, Andrea; Gupta, Tripta; Bennett, Jeffrey L; Verkman, A S

    2011-05-01

    Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity. PMID:21454592

  14. Residue 21 of human granulocyte-macrophage colony-stimulating factor is critical for biological activity and for high but not low affinity binding.

    OpenAIRE

    Lopez, A F; Shannon, M F; Hercus, T; Nicola, N A; Cambareri, B; Dottore, M; Layton, M. J.; Eglinton, L; Vadas, M A

    1992-01-01

    The functional role of the predicted first alpha-helix of human granulocyte-macrophage colony-stimulating factor (GM-CSF) was analysed by site-directed mutagenesis and multiple biological and receptor binding assays. Initial deletion mutagenesis pointed to residues 20 and 21 being critical. Substitution mutagenesis showed that by altering Gln20 to Ala full GM-CSF activity was retained but that by altering Glu21 for Ala GM-CSF activity and high affinity receptor binding were decreased. Substit...

  15. Knowledge-based fragment binding prediction.

    Science.gov (United States)

    Tang, Grace W; Altman, Russ B

    2014-04-01

    Target-based drug discovery must assess many drug-like compounds for potential activity. Focusing on low-molecular-weight compounds (fragments) can dramatically reduce the chemical search space. However, approaches for determining protein-fragment interactions have limitations. Experimental assays are time-consuming, expensive, and not always applicable. At the same time, computational approaches using physics-based methods have limited accuracy. With increasing high-resolution structural data for protein-ligand complexes, there is now an opportunity for data-driven approaches to fragment binding prediction. We present FragFEATURE, a machine learning approach to predict small molecule fragments preferred by a target protein structure. We first create a knowledge base of protein structural environments annotated with the small molecule substructures they bind. These substructures have low-molecular weight and serve as a proxy for fragments. FragFEATURE then compares the structural environments within a target protein to those in the knowledge base to retrieve statistically preferred fragments. It merges information across diverse ligands with shared substructures to generate predictions. Our results demonstrate FragFEATURE's ability to rediscover fragments corresponding to the ligand bound with 74% precision and 82% recall on average. For many protein targets, it identifies high scoring fragments that are substructures of known inhibitors. FragFEATURE thus predicts fragments that can serve as inputs to fragment-based drug design or serve as refinement criteria for creating target-specific compound libraries for experimental or computational screening. PMID:24762971

  16. Identification of High Affinity Fatty Acid Binding Sites on Human Serum Albumin by MM-PBSA Method

    OpenAIRE

    Fujiwara, Shin-ichi; Amisaki, Takashi

    2007-01-01

    Human serum albumin (HSA) has seven common fatty acid (FA) binding sites. In this study, we used the molecular mechanics Poisson-Boltzmann surface area method to identify high affinity FA binding sites on HSA in terms of binding free energy. Using multiple HSA-FA (myristate, palmitate) complex models constructed by molecular dynamics simulations, two methods were performed in molecular mechanics Poisson-Boltzmann surface area, the “three-trajectory method” and the “single-trajectory method”. ...

  17. Computational Estimates of Binding Affinities for Estrogen Receptor Isoforms in Rainbow Trout

    CERN Document Server

    Shyu, Conrad

    2009-01-01

    Molecular dynamics simulations are performed to determine the binding affinities between the hormone 17 beta-estradiol (E2) and different estrogen receptor (ER) isoforms in the rainbow trout (Oncorhynchus mykiss). Previous studies have demonstrated that a recent, unique gene duplication of the ER alpha subtype created two isoforms ER alpha 1 and ER alpha 2, and an early secondary split of ER beta produced two distinct isoforms of ER beta 1 and ER beta 2 based on the phylogenetic analysis. The objective of our computational studies is to provide insight into the underlying evolutionary selection pressure on the ER isoforms. Our results show that E2 binds preferentially to ER alpha 1. This finding corresponds to the experimental results as the ERs evolved from gene duplication events are frequently free from selective pressure and should exhibit no deleterious effects. The E2, however, only binds slightly better to ER beta 2. Both isoforms remain competitive. This finding reflects the fact that since ER beta 2 ...

  18. Interleukin 3 activates human blood basophils via high-affinity binding sites

    International Nuclear Information System (INIS)

    Pure populations of human basophilic granulocytes were obtained from chronic myeloid leukemia (CML) blood by negative selection using a mixture of monoclonal antibodies and complement. 125I-radiolabeled recombinant human interleukin 3 (rhIL-3) bound to purified basophils in a specific manner. Quantitative binding studies and Scatchard plot analyses performed on samples from two donors revealed the presence of a single class of high-affinity IL-3 binding sites. Purified CML basophils maintained in suspension in the presence of rhIL-3 incorporated up to 12 times more [3H]thymidine than basophils in control cultures. Furthermore, after preincubation in vitro with rhIL-3 for 30 min, normal blood basophils released 2- to 3-fold more histamine than basophils pretreated with control medium when exposed to various concentrations of an anti-IgE antibody. Together, these results show that rhIL-3 binds to a specific receptor on blood basophils and is a regulator of basophil function

  19. Ligand-Binding Affinity Estimates Supported by Quantum-Mechanical Methods.

    Science.gov (United States)

    Ryde, Ulf; Söderhjelm, Pär

    2016-05-11

    One of the largest challenges of computational chemistry is calculation of accurate free energies for the binding of a small molecule to a biological macromolecule, which has immense implications in drug development. It is well-known that standard molecular-mechanics force fields used in most such calculations have a limited accuracy. Therefore, there has been a great interest in improving the estimates using quantum-mechanical (QM) methods. We review here approaches involving explicit QM energies to calculate binding affinities, with an emphasis on the methods, rather than on specific applications. Many different QM methods have been employed, ranging from semiempirical QM calculations, via density-functional theory, to strict coupled-cluster calculations. Dispersion and other empirical corrections are mandatory for the approximate methods, as well as large basis sets for the stricter methods. QM has been used for the ligand, for a few crucial groups around the ligand, for all the closest atoms (200-1000 atoms), or for the full receptor-ligand complex, but it is likely that with a proper embedding it might be enough to include all groups within ∼6 Å of the ligand. Approaches involving minimized structures, simulations of the end states of the binding reaction, or full free-energy simulations have been tested. PMID:27077817

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

    International Nuclear Information System (INIS)

    FMRFamide (Phe-Met-Arg-Phe-NH2) 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 3[H]-dihydromorphine and 3[H]-ethylketocyclazocine (IC50 = 14 μM and 320 μM, 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

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

    International Nuclear Information System (INIS)

    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 125I-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

  2. Enhancement of the binding affinity of methylene blue to site I in human serum albumin by cupric and ferric ions.

    Science.gov (United States)

    He, Ling-Ling; Wang, Yong-Xia; Wu, Xiao-Xia; Liu, Xian-Ping; Wang, Xin; Liu, Bin; Wang, Xin

    2015-12-01

    In this work, the binding characteristics of methylene blue (MB) to human serum albumin (HSA) and the influence of Cu(2+) and Fe(3+) on the binding affinity of MB to HSA were investigated using fluorescence, absorption, circular dichroism (CD) spectroscopy and molecular modelling. The results of competitive binding experiments using the site probes ketoprofen and ibuprofen as specific markers suggested that MB was located in site I within sub-domain IIA of HSA. The molecular modelling results agreed with the results of competitive site marker experiments and the results of CD spectra indicated that the interaction between MB and HSA caused the conformational changes in HSA. The binding affinity of MB to HSA was enhanced but to a different extent in the presence of Cu(2+) and Fe(3+), respectively, which indicated that the influence of different metal ions varied. Enhancement of the binding affinity of MB to HSA in the presence of Cu(2+) is due to the formation of Cu(2+)-HSA complex leading to the conformational changes in HSA, whereas in the presence of Fe(3+), enhancement of the binding affinity is due to the greater stability of the Fe(3+)-HSA-MB complex compared with the MB-HSA complex. PMID:25833007

  3. Introduction of D-Phenylalanine enhanced the receptor binding affinities of gonadotropin-releasing hormone peptides

    OpenAIRE

    Lu, Jie; Hathaway, Helen J.; Royce, Melanie E.; Prossnitz, Eric R.; Miao, Yubin

    2014-01-01

    The purpose of this study was to examine whether the introduction of D-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated D-Lys6-GnRH peptides. Building upon the construct of DOTA-Ahx-(D-Lys6-GnRH1) we previously reported, an aromatic amino acid of D-Phe was inserted either between the DOTA and Ahx or between the Ahx and D-Lys6 to generate new DOTA-D-Phe-Ahx-(D-Lys6-GnRH) or DOTA-Ahx-D-Phe-(D-Lys6-GnRH) peptides. Compared to DOTA-Ahx-(D-Lys6-GnRH1) (36.1 nM), the introd...

  4. Localization of the binding site for the human high-affinity Fc receptor on IgG.

    Science.gov (United States)

    Duncan, A R; Woof, J M; Partridge, L J; Burton, D R; Winter, G

    1988-04-01

    A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen. PMID:2965792

  5. Molecular dynamic behavior and binding affinity of flavonoid analogues to the cyclin dependent kinase 6/cyclin D complex.

    Science.gov (United States)

    Khuntawee, Wasinee; Rungrotmongkol, Thanyada; Hannongbua, Supot

    2012-01-23

    The cyclin dependent kinases (CDKs), each with their respective regulatory partner cyclin that are involved in the regulation of the cell cycle, apoptosis, and transcription, are potentially interesting targets for cancer therapy. The CDK6 complex with cyclin D (CDK6/cycD) drives cellular proliferation by phosphorylation of specific key target proteins. To understand the flavonoids that inhibit the CDK6/cycD functions, molecular dynamics simulations (MDSs) were performed on three inhibitors, fisetin (FST), apigenin (AGN), and chrysin (CHS), complexed with CDK6/cycD, including the two different binding orientations of CHS: FST-like (CHS_A) and deschloro-flavopiridol-like (CHS_B). For all three inhibitors, including both CHS orientations, the conserved interaction between the 4-keto group of the flavonoid and the backbone V101 nitrogen of CDK6 was strongly detected. The 3'- and 4'-OH groups on the flavonoid phenyl ring and the 3-OH group on the benzopyranone ring of inhibitor were found to significantly increase the binding and inhibitory efficiency. Besides the electrostatic interactions, especially through hydrogen bond formation, the van der Waals (vdW) interactions with the I19, V27, F98, H100, and L152 residues of CDK6 are also important factors in the binding efficiency of flavonoids against the CDK6/cycD complex. On the basis of the docking calculation and MM-PBSA method, the order of the predicted inhibitory affinities of these three inhibitors toward the CDK6/cycD was FST > AGN > CHS, which is in good agreement with the experimental data. In addition, CHS preferentially binds to the active CDK6 in a different orientation to FST and AGN but similar to its related analog, deschloro-flavopiridol. The obtained results are useful as the basic information for the further design of potent anticancer drugs specifically targeting the CDK6 enzyme. PMID:22172011

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

    Science.gov (United States)

    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

  7. Fluorescence Characterization of Dissolved Organic Matter Isolates from Sediments and the Association with Phenanthrene Binding Affinity

    Science.gov (United States)

    Hur, Jin; Lee, Bo-Mi; Shin, Kyung-Hoon

    2014-05-01

    In this study, selected spectroscopic characteristics of sediment organic matter (SOM) were compared and discussed with respect to their different isolation methods, the source discrimination capabilities, and the association with the extent of phenanthrene binding. A total of 16 sediments were collected from three categorized locations including a costal lake, industrial areas, and the upper streams, each of which is likely influenced by the organic sources of algal production, industrial effluent, and terrestrial input, respectively. The spectroscopic properties related to aromatic structures and terrestrial humic acids were more pronounced for alkaline extractable organic matter (AEOM) isolates than for the SOM isolates based on water soluble extracts and porewater. The three categorized sampling locations were the most differentiated in the AEOM isolates, suggesting AEOM may be the most representative SOM isolates in describing the chemical properties and the organic sources of SOM. Parallel factor analysis (PARAFAC) based on fluorescence excitation-emission matrix (EEM) showed that a combination of four fluorescent groups could represent all the fluorescence features of SOM. The three categorized sampling locations were well discriminated by the percent distributions of terrestrial and microbial humic-like fluorescent groups of the AEOM isolates. The relative distribution of terrestrial humic-like fluorophores was highly correlated with the extent of phenanthrene binding (r=0.676; phumic acids in SOM may contribute to the enhancement of binding with hydrophobic organic contaminants in sediments. Principal component analysis (PCA) further demonstrated that the extent of SOM's binding affinity might be affected by the degree of biological transformation in SOM as well as the abundance of aromatic carbon structures.

  8. Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus embryos

    Directory of Open Access Journals (Sweden)

    Bogdanović Ozren

    2011-08-01

    Full Text Available Abstract Background DNA methylation is a widespread epigenetic modification in vertebrate genomes. Genomic sites of DNA methylation can be bound by methyl-CpG-binding domain proteins (MBDs and specific zinc finger proteins, which can recruit co-repressor complexes to silence transcription on targeted loci. The binding to methylated DNA may be regulated by post-translational MBD modifications. Findings A methylated DNA affinity precipitation method was implemented to assay binding of proteins to methylated DNA. Endogenous MeCP2 and MBD3 were precipitated from Xenopus oocyte extracts and conditions for methylation-specific binding were optimized. For a reverse experiment, DNA methylation in early Xenopus embryos was assessed by MBD affinity capture. Conclusions A methylated DNA affinity resin can be applied to probe for MBD activity in extracts. This assay has a broad application potential as it can be coupled to downstream procedures such as western blotting, fluorimetric HDAC assays and quantitative mass spectrometry. Methylated DNA affinity capture by methyl-CpG binding proteins produces fractions highly enriched for methylated DNA, suitable for coupling to next generation sequencing technologies. The two enrichment strategies allow probing of methyl-CpG protein interactions in early vertebrate oocytes and embryos.

  9. Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

    Science.gov (United States)

    Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

    2016-10-01

    Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. PMID:27371890

  10. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin

    Directory of Open Access Journals (Sweden)

    Chung Chun-wa

    2011-04-01

    Full Text Available Abstract Background Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA. It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. Results We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which - in drugs sites 1 and 2 on the protein - are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. Conclusions The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.

  11. Distinction between high-affinity [3H]phencyclidine binding sites and muscarinic receptors in guinea-pig ileum muscle

    International Nuclear Information System (INIS)

    [3H]Phencyclidine ([3H]PCP) binding was studied in guinea-pig ileum muscle membranes. Specific binding of [3H]PCP was time dependent, reversible and saturable, with an equilibrium dissociation constant of 154 nM and maximum binding of 12.9 pmol/mg of protein at pH 9. Its pH dependency suggests that the un-ionized PCP is the pharmacologically active form. The binding site was on a protein which was sensitive to heat, proteolytic enzymes and the carboxylic group reagent dicyclohexylcarbodiimide, but insensitive to phospholipase A and C, concanavalin A, dithiothreitol and N-ethylmaleimide. Specific [3H]PCP binding was displaced effectively by several PCP analogs and Ca++ channel antagonists including verapamil, to which these sites had a high affinity. These high-affinity PCP-binding sites were found at a much higher concentration in the same membrane preparation than muscarinic receptor sites identified by their specific binding of [3H]quinuclidinyl benzilate. PCP bound to both sites, but with a lower affinity to the muscarinic receptor sites. The PCP and muscarinic receptor sites differed in their sensitivities to pH and drug specifities

  12. NetMHCpan, a method for MHC class I binding prediction beyond humans

    DEFF Research Database (Denmark)

    Hoof, Ilka; Peters, B; Sidney, J;

    2009-01-01

    Binding of peptides to major histocompatibility complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC genomic region (called HLA) is extremely polymorphic comprising several thousand alleles, each encoding a distinct...... method that generates quantitative predictions of the affinity of any peptide-MHC class I interaction. NetMHCpan-2.0 has been trained on the hitherto largest set of quantitative MHC binding data available, covering HLA-A and HLA-B, as well as chimpanzee, rhesus macaque, gorilla, and mouse MHC class I...... molecules. We show that the NetMHCpan-2.0 method can accurately predict binding to uncharacterized HLA molecules, including HLA-C and HLA-G. Moreover, NetMHCpan-2.0 is demonstrated to accurately predict peptide binding to chimpanzee and macaque MHC class I molecules. The power of NetMHCpan-2.0 to guide...

  13. High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK

    OpenAIRE

    Schloot Nanette C; Fingberg Waltraud; Alloza Iraide; Vandenbroeck Koen; Blasius Elias; Siegenthaler Rahel K; Burkart Volker; Christen Philipp; Kolb Hubert

    2010-01-01

    Abstract Background Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. Results Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further regi...

  14. Whole blood-oxygen binding properties of four cold-temperate marine fishes: blood affinity is independent of pH-dependent binding, routine swimming performance, and environmental hypoxia

    DEFF Research Database (Denmark)

    Herbert, Neill A; Skov, Peter V; Wells, Rufus M G;

    2006-01-01

    The relationship between whole blood-oxygen affinity (P(50)) and pH-dependent binding (i.e., cooperativity and the Bohr [ Phi ] and Root effects) was examined statistically under standardized conditions (10.0 degrees Celsius) in four unrelated cold-temperate marine fishes that differ widely in...... swimming performance and the predicted low O(2) response of each species. The ecotype of the four marine species was also unrelated to pH-dependent binding because no difference in the Bohr effect was apparent ( Phi varied insignificantly from -0.90 to -1.06), and differences in the magnitude of the...... their swimming performance and their expected responses to hypoxia: cod (Gadus morhua), herring (Clupea harengus), mackerel (Scomber scombrus), and plaice (Pleuronectes platessa). An unexpected difference in blood-oxygen affinity was found (herring>plaice>mackerel>cod), and this was independent of both...

  15. Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines.

    Science.gov (United States)

    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

  16. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R;

    2005-01-01

    molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have...

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

    DEFF Research Database (Denmark)

    Vogensen, Stine B.; Marek, Ales; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin Holst Friborg; 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. Screen...

  18. A community resource benchmarking predictions of peptide binding to MHC-I molecules

    DEFF Research Database (Denmark)

    Peters, B; Bui, HH; Pletscher-Frankild, Sune;

    2006-01-01

    entire pathogen proteomes for peptide likely to bind MHC. Here we make public a large set of 48,828 quantitative peptide-binding affinity measurements relating to 48 different mouse, human, macaque, and chimpanzee MHC class I alleles. We use this data to establish a set of benchmark predictions with one...... neural network method and two matrix-based prediction methods extensively utilized in our groups. In general, the neural network outperforms the matrix-based predictions mainly due to its ability to generalize even on a small amount of data. We also retrieved predictions from tools publicly available on...... the internet. While differences in the data used to generate these predictions hamper direct comparisons, we do conclude that tools based on combinatorial peptide libraries perform remarkably well. The transparent prediction evaluation on this dataset provides tool developers with a benchmark for...

  19. SYNTHESIS OF POLY(3,4-AZOPYRIDYLENE) AND OXYGEN-BINDING AFFINITY OF ITS COMPLEX WITH COBALTPORPHYRIN

    Institute of Scientific and Technical Information of China (English)

    Bao-qing Shentu; Zhi-xue Weng; Hiroyuki Nishide

    2005-01-01

    Synthesis and characteristics of poly(3,4-azopyridylene) (PAP), conductivity and oxygen-binding affinity of its complex with meso-α,α,α,α-tetrakis(o-pivalamidophenyl) porphyrinatocobalt(Ⅱ) (CoP) were studied. PAP was prepared by oxidative polymerization of 3,4-diaminopyridine (DAP) in DMF solution using CuCl/pyridine as the catalyst. IR and NMR results showed that the peak of amido group in DAP was converted to the azo group in PAP and a π conjugated polymer was synthesized. The average molecular weight of PAP was determined to be 5.0 × 103. The PAP-CoP complex was prepared by complexing the pyridyl group of PAP with the fifth coordination site of CoP in DMF solution. In comparison with the CoP complex with a non-π conjugated polymer, the PAP-CoP complex shows good electroconductivity of 5.8 × 10-6 Scm-1. The PAP-CoP complex displays a reversible change in the UV-Visible absorption spectrum from the deoxy form to the oxy or oxygen-binding one with an isosbestic point, in response to the partial oxygen pressure of the atmosphere. The oxygen-response behavior was monitored at the absorbance ascribed to the oxy form at 548 nm to give the oxygen-binding affinity.The oxygen-binding equilibrium curves of PAP-CoP complex obey a Langmuir isotherm. DMF has great effects on the oxygen-binding properties of the PAP-CoP complex. The oxygen-binding affinity of PAP-CoP complex in the solid state is higher than that in DMF solution. With decreasing temperature, the oxygen-binding affinity of the PAP-CoP complex increases.

  20. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    International Nuclear Information System (INIS)

    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 [125I]EDP I, [125I]Glu-plasminogen, and [125I]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 [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, 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. α2-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 [125I]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

  1. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    Science.gov (United States)

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-01

    Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids. PMID:21797258

  2. SAAMBE: Webserver to Predict the Charge of Binding Free Energy Caused by Amino Acids Mutations

    OpenAIRE

    Marharyta Petukh; Luogeng Dai; Emil Alexov

    2016-01-01

    Predicting the effect of amino acid substitutions on protein–protein affinity (typically evaluated via the change of protein binding free energy) is important for both understanding the disease-causing mechanism of missense mutations and guiding protein engineering. In addition, researchers are also interested in understanding which energy components are mostly affected by the mutation and how the mutation affects the overall structure of the corresponding protein. Here we report a webserver,...

  3. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Science.gov (United States)

    Asti, Lorenzo; Uguzzoni, Guido; Marcatili, Paolo; Pagnani, Andrea

    2016-04-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6), outperforming other sequence- and structure-based models. PMID:27074145

  4. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity

    Science.gov (United States)

    Marcatili, Paolo; Pagnani, Andrea

    2016-01-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10−6), outperforming other sequence- and structure-based models. PMID:27074145

  5. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Directory of Open Access Journals (Sweden)

    Lorenzo Asti

    2016-04-01

    Full Text Available The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6, outperforming other sequence- and structure-based models.

  6. Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism

    OpenAIRE

    Bourne, Yves; Radić, Zoran; Aráoz, Rómulo; Talley, Todd T.; Benoit, Evelyne; Servent, Denis; Taylor, Palmer; Molgó, Jordi; Marchot, Pascale

    2010-01-01

    Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type α12βγδ and neuronal α3β2 and α4β2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble rec...

  7. A Self-Adaptive Steered Molecular Dynamics Method Based on Minimization of Stretching Force Reveals the Binding Affinity of Protein–Ligand Complexes

    Directory of Open Access Journals (Sweden)

    Junfeng Gu

    2015-10-01

    Full Text Available Binding affinity prediction of protein–ligand complexes has attracted widespread interest. In this study, a self-adaptive steered molecular dynamics (SMD method is proposed to reveal the binding affinity of protein–ligand complexes. The SMD method is executed through adjusting pulling direction to find an optimum trajectory of ligand dissociation, which is realized by minimizing the stretching force automatically. The SMD method is then used to simulate the dissociations of 19 common protein–ligand complexes which are derived from two homology families, and the binding free energy values are gained through experimental techniques. Results show that the proposed SMD method follows a different dissociation pathway with lower a rupture force and energy barrier when compared with the conventional SMD method, and further analysis indicates the rupture forces of the complexes in the same protein family correlate well with their binding free energy, which reveals the possibility of using the proposed SMD method to identify the active ligand.

  8. Developments of segregation process and chemical analysis for virus receptor recognizing environmental signal substance by affinity binding assaying

    International Nuclear Information System (INIS)

    Virus receptors recognizing environmental signal were segregated and analyzed chemically by affinity binding assaying using radio-isotope marked compound. Pathogenic protein in rice stem virus was indicated possibility of a protein which participates in transfer process between the virus cells. An enzyme, Chitobiose was separated from a bacteria, actinomycete. It was cleared by 3H chitobiose affinity binding assaying that two kinds of chitobiose binding protein existed in cell membrane of the actinomycete. A different kind of protein was founded in the cell membrane which was raised in existence of chitobiose. Observation showed that the protein was different from a protein in the cell membrane which was raised in non-existence of chitobiose. These two kinds of protein might be formed a complex compound on the surface of cell membrane. (M. Suetake)

  9. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lund, Ole

    2009-01-01

    this binding event. RESULTS: Here, we present a novel artificial neural network-based method, NN-align that allows for simultaneous identification of the MHC class II binding core and binding affinity. NN-align is trained using a novel training algorithm that allows for correction of bias in the...... training data due to redundant binding core representation. Incorporation of information about the residues flanking the peptide-binding core is shown to significantly improve the prediction accuracy. The method is evaluated on a large-scale benchmark consisting of six independent data sets covering 14...

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

    Science.gov (United States)

    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. Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

    Science.gov (United States)

    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. High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK

    Directory of Open Access Journals (Sweden)

    Schloot Nanette C

    2010-11-01

    Full Text Available Abstract Background Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (preproinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. Results Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further region in each proinsulin domain (A- and B-chain, C-peptide. Among the latter, peptides covering most of the B-chain region B11-23 exhibited strongest binding, which was in the range of known high-affinity DnaK ligands, dissociation equilibrium constant (K'd of 2.2 ± 0.4 μM. The B-chain region B11-23 is located at the interface between two insulin molecules and not accessible in insulin oligomers. Indeed, native insulin oligomers showed very low DnaK affinity (K'd 67.8 ± 20.8 μM whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM. Conclusions Intact insulin only weakly interacts with the hsp70 chaperone DnaK whereas monomeric proinsulin and peptides from 3 distinct proinsulin regions show substantial chaperone binding. Strongest binding was seen for the B-chain peptide B 11-23. Interestingly, peptide B11-23 represents a dominant autoantigen in type 1 diabetes.

  13. Binding affinities in the SAMPL3 trypsin and host-guest blind tests estimated with the MM/PBSA and LIE methods.

    OpenAIRE

    Mikulskis, Paulius; Genheden, Samuel; Rydberg, Patrik; Sandberg, Lars; Olsen, Lars; Ryde, Ulf

    2012-01-01

    We have estimated affinities for the binding of 34 ligands to trypsin and nine guest molecules to three different hosts in the SAMPL3 blind challenge, using the MM/PBSA, MM/GBSA, LIE, continuum LIE, and Glide score methods. For the trypsin challenge, none of the methods were able to accurately predict the experimental results. For the MM/GB(PB)SA and LIE methods, the rankings were essentially random and the mean absolute deviations were much worse than a null hypothesis giving the same affini...

  14. Proliferative responses and binding properties of hematopoietic cells transfected with low-affinity receptors for leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor.

    OpenAIRE

    Gearing, D P; Ziegler, S F; Comeau, M R; Friend, D; Thoma, B; Cosman, D; Park, L.; Mosley, B

    1994-01-01

    Specific low-affinity receptors for leukemia inhibitory factor (LIF), oncostatin M (OSM; gp130), and ciliary neurotrophic factor (CNTF; receptor alpha, CNTFR alpha) may be utilized in various combinations to generate high-affinity binding sites and signal transduction. We have tested the ability of combinations of these receptors to transduce a proliferative signal in BAF-B03 cells. Coexpression of the LIF receptor and gp130 in these cells conferred high-affinity LIF and OSM binding and respo...

  15. Design of Bcl-2 and Bcl-xL Inhibitors with Subnanomolar Binding Affinities Based upon a New Scaffold

    Energy Technology Data Exchange (ETDEWEB)

    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 (Michigan)

    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.

  16. Comparison of high affinity binding of 3H-proadifen and 3H-(-)-cocaine t rat liver membranes

    International Nuclear Information System (INIS)

    The characteristics of the binding of 3H-proadifen to rat liver membranes were studied and compared to those of 3H-cocaine. It was found that 3H-proadifen was bound reversibly with high affinity (KD=1.8±0.5 nM) and large capacity (Bmax=2010±340 pmol/g wet tissue) to liver membranes. The corresponding values for the 3H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of 3H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 μM CdCl2 in the incubation buffer it was possible to differentiate between two 3H-cocaine binding sites with Kd values of 1.6 and 7.7 nM and Bmax values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of 3H-proadifen and 3H-cocaine inhibited the binding of 3H-proadifen (IC50=10 nM) and proadifen that of 3H-cocaine (IC50=1 nM). There was a high correlation coefficient (rr=0.972; P50=100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl2, ZnCl2 and CuCl2 inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd2+ on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15μM. The similarity of the characteristics of the binding of these ligands with that of 3H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.)

  17. Molecular Weight, Protein Binding Affinity and Methane Mitigation of Condensed Tannins from Mangosteen-peel (Garcinia mangostana L).

    Science.gov (United States)

    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. PMID:26323400

  18. Syntheses of 7-Substituted α-Cyperone Derivatives for Selective Sigma-1 Receptor over Cannabinoid-1 Receptor Binding Affinities

    International Nuclear Information System (INIS)

    We have successfully synthesized seven α-cyperone derivatives and found that the presence of a hydrogen bond donor/acceptor groups at the C7 position of α-cyperone significantly affects specificity and potency of CB1 receptor binding affinity over sigma-1 receptor binding affinity. In particular, the presence of the amino moiety at the C7 position of α-cyperone is beneficial for binding to sigmia-1 receptor. The molecular mechanism of compound 8 involved in the high binding affinity to sigma-1 receptor is under investigation. We first synthesized α-cyperone 1 by following the previously reported synthetic routes.15-19 In brief, azeotropic imination of (+)-dihydrocarvone and (R)-(+)-1-phenylethylamine followed by alkylation with a slight excess of ethyl vinyl ketone (EVK) in THF at 40 .deg. C produced the Micheal adduct. The resulting adduct was hydrolyzed and then treated with sodium methoxide at room temperature to give an easily separable mixture of α-cyperone 1 and its side product. Flash chromatography resulted in pure α-cyperone 1 in a 30% yield from (+)-dihydrocarvone

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

    Science.gov (United States)

    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

  20. Predicting peptides binding to MHC class II molecules using multi-objective evolutionary algorithms

    Directory of Open Access Journals (Sweden)

    Feng Lin

    2007-11-01

    Full Text Available Abstract Background Peptides binding to Major Histocompatibility Complex (MHC class II molecules are crucial for initiation and regulation of immune responses. Predicting peptides that bind to a specific MHC molecule plays an important role in determining potential candidates for vaccines. The binding groove in class II MHC is open at both ends, allowing peptides longer than 9-mer to bind. Finding the consensus motif facilitating the binding of peptides to a MHC class II molecule is difficult because of different lengths of binding peptides and varying location of 9-mer binding core. The level of difficulty increases when the molecule is promiscuous and binds to a large number of low affinity peptides. In this paper, we propose two approaches using multi-objective evolutionary algorithms (MOEA for predicting peptides binding to MHC class II molecules. One uses the information from both binders and non-binders for self-discovery of motifs. The other, in addition, uses information from experimentally determined motifs for guided-discovery of motifs. Results The proposed methods are intended for finding peptides binding to MHC class II I-Ag7 molecule – a promiscuous binder to a large number of low affinity peptides. Cross-validation results across experiments on two motifs derived for I-Ag7 datasets demonstrate better generalization abilities and accuracies of the present method over earlier approaches. Further, the proposed method was validated and compared on two publicly available benchmark datasets: (1 an ensemble of qualitative HLA-DRB1*0401 peptide data obtained from five different sources, and (2 quantitative peptide data obtained for sixteen different alleles comprising of three mouse alleles and thirteen HLA alleles. The proposed method outperformed earlier methods on most datasets, indicating that it is well suited for finding peptides binding to MHC class II molecules. Conclusion We present two MOEA-based algorithms for finding motifs

  1. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity

    DEFF Research Database (Denmark)

    Asti, Lorenzo; Uguzzoni, Guido; Marcatili, Paolo;

    2016-01-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high...... HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6), outperforming other sequence- and structure-based models....

  2. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency.

    Science.gov (United States)

    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

  3. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency

    Science.gov (United States)

    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

  4. Predicted metal binding sites for phytoremediation

    OpenAIRE

    Sharma, Ashok; Roy, Sudeep; Tripathi, Kumar Parijat; Roy, Pratibha; Mishra, Manoj; Khan, Feroz; Meena, Abha

    2009-01-01

    Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. The objective of the study is to design and analyze metal binding motifs against the genes involved in phytoremediation. This is being done on the basis of certain pre-requisite amino-acid residues known to bind metal ions/metal complexes in medicinal and aromatic plants (MAP's). Earlier work on MAP's have shown that heavy metals accumulated by aromatic and medicinal plants do no...

  5. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins.

    Science.gov (United States)

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-04-19

    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

  6. Irreversible blockade of the high and low affinity (3H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

    International Nuclear Information System (INIS)

    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 (3H)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+-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of (3H)naloxone, whereas the dihydrocodeinone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested

  7. Irreversible blockade of the high and low affinity ( sup 3 H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

    Energy Technology Data Exchange (ETDEWEB)

    Krizsan, D. (EGIS Pharmaceutical Works, Budapest (Hungary)); Varga, E.; Benyhe, S.; Szucs, M.; Borsodi, A. (Biological Research Center of the Hungarian Academy of Sciences, Szeged (Hungary)); Hosztafi, S. (Alkaloida Chemical Works, Tiszavasvari (Hungary))

    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.

  8. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Feng; Miyakawa, Takuya [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kataoka, Michihiko [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Takeshita, Daijiro [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kumashiro, Shoko [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Uzura, Atsuko [Research and Development Center, Nagase and Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241 (Japan); Urano, Nobuyuki [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Nagata, Koji [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shimizu, Sakayu [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    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.

  9. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    International Nuclear Information System (INIS)

    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

  10. Assessing the binding affinity of a selected class of DPP4 inhibitors using chemical descriptor-based multiple linear regression

    OpenAIRE

    Jose Isagani Janairo; Gerardo Janairo; Frumencio Co; Derrick Ethelbhert Yu

    2011-01-01

    The activity of a selected class of DPP4 inhibitors was preliminarily assessed using chemical descriptors derived AM1 optimized geometries. Using multiple linear regression model, it was found that ?E0, LUMO energy, area, molecular weight and ?H0 are the significant descriptors that can adequately assess the binding affinity of the compounds. The derived multiple linear regression (MLR) model was validated using rigorous statistical analysis. The preliminary model suggests t...

  11. Estimation of apparent binding constant of complexes of selected acyclic nucleoside phosphonates with -cyclodextrin by affinity capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Šolínová, Veronika; Mikysková, Hana; Kaiser, Martin Maxmilian; Janeba, Zlatko; Holý, Antonín; Kašička, Václav

    2016-01-01

    Roč. 37, č. 2 (2016), s. 239-247. ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA13-17224S; GA ČR(CZ) GA15-01948S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * affinity capillary electrophoresis * binding constant * nucleotide analogs * beta-cyclodextrin Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.028, year: 2014

  12. Binding Affinity and Specificity of Neuromyelitis Optica Autoantibodies to Aquaporin-4 M1/M23 Isoforms and Orthogonal Arrays*

    OpenAIRE

    Crane, Jonathan M.; Lam, Chiwah; Rossi, Andrea; Gupta, Tripta; Bennett, Jeffrey L.; Verkman, A S

    2011-01-01

    Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-r...

  13. Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled Protein A Caused by Affinity Binding with Immunoglobulin G in Bovine Plasma

    Directory of Open Access Journals (Sweden)

    Kiyotaka Sakai

    2009-10-01

    Full Text Available Fluorescence enhancement of fluorescein isothiocyanate-labeled protein A (FITC-protein A caused by the binding with immunoglobulin G (IgG in bovine plasma was studied. FITC-protein A was immobilized onto a glass surface by covalent bonds. An increase in fluorescence intensity was dependent on IgG concentration ranging from 20 to 78 μg/mL in both phosphate buffer saline and bovine plasma. This method requires no separation procedure, and the reaction time is less than 15 min. A fluorescence enhancement assay by the affinity binding of fluorescence-labeled reagent is thus available for the rapid determination of biomolecules in plasma.

  14. High- and low-affinity binding of S-citalopram to the human serotonin transporter mutated at 20 putatively important amino acid positions

    DEFF Research Database (Denmark)

    Plenge, Per; Wiborg, Ove

    2005-01-01

    uptake inhibitors and serotonin (5-HT) have been found on SERT. At one site, uptake inhibitors bind with high-affinity to SERT, thereby blocking the uptake of 5-HT. The other site is a low-affinity allosteric site, which influences the dissociation of uptake inhibitors, such as imipramine, paroxetine......, and citalopram from the first site, when occupied by 5-HT and a few uptake inhibitors like paroxetine and citalopram. In this study, the connection between the high-affinity binding site and the allosteric affinity-modulating site was investigated by introducing 20 single amino acid substitutions into...

  15. Predicting Adsorption Affinities of Small Molecules on Carbon Nanotubes Using Molecular Dynamics Simulation.

    Science.gov (United States)

    Comer, Jeffrey; Chen, Ran; Poblete, Horacio; Vergara-Jaque, Ariela; Riviere, Jim E

    2015-12-22

    Computational techniques have the potential to accelerate the design and optimization of nanomaterials for applications such as drug delivery and contaminant removal; however, the success of such techniques requires reliable models of nanomaterial surfaces as well as accurate descriptions of their interactions with relevant solutes. In the present work, we evaluate the ability of selected models of naked and hydroxylated carbon nanotubes to predict adsorption equilibrium constants for about 30 small aromatic compounds with a variety of functional groups. The equilibrium constants determined using molecular dynamics coupled with free-energy calculation techniques are directly compared to those derived from experimental measurements. The calculations are highly predictive of the relative adsorption affinities of the compounds, with excellent correlation (r ≥ 0.9) between calculated and measured values of the logarithm of the adsorption equilibrium constant. Moreover, the agreement in absolute terms is also reasonable, with average errors of less than one decade. We also explore possible effects of surface loading, although we demonstrate that they are negligible for the experimental conditions considered. Given the degree of reliability demonstrated, we move on to employing the in silico techniques in the design of nanomaterials, using the optimization of adsorption affinity for the herbacide atrazine as an example. Our simulations suggest that, compared to other modifications of graphenic carbon, polyvinylpyrrolidone conjugation gives the highest affinity for atrazine-substantially greater than that of graphenic carbon alone-and may be useful as a nanomaterial for delivery or sequestration of atrazine. PMID:26506132

  16. Structural Changes in the Lectin Domain of CD23, the Low-Affinity IgE Receptor, upon Calcium Binding

    Energy Technology Data Exchange (ETDEWEB)

    Wurzburg, Beth A.; Tarchevskaya, Svetlana S.; Jardetzky, Theodore S. (NWU)

    2010-03-08

    CD23, the low-affinity receptor for IgE (Fc{var_epsilon}RII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca{sup 2+}. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca{sup 2+} bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.

  17. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.

    Science.gov (United States)

    Fischer, Marcus; Hopkins, Adam P; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G; Hubbard, Roderick E; Thomas, Gavin H

    2015-11-01

    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

  18. SAAMBE: Webserver to Predict the Charge of Binding Free Energy Caused by Amino Acids Mutations

    Directory of Open Access Journals (Sweden)

    Marharyta Petukh

    2016-04-01

    Full Text Available Predicting the effect of amino acid substitutions on protein–protein affinity (typically evaluated via the change of protein binding free energy is important for both understanding the disease-causing mechanism of missense mutations and guiding protein engineering. In addition, researchers are also interested in understanding which energy components are mostly affected by the mutation and how the mutation affects the overall structure of the corresponding protein. Here we report a webserver, the Single Amino Acid Mutation based change in Binding free Energy (SAAMBE webserver, which addresses the demand for tools for predicting the change of protein binding free energy. SAAMBE is an easy to use webserver, which only requires that a coordinate file be inputted and the user is provided with various, but easy to navigate, options. The user specifies the mutation position, wild type residue and type of mutation to be made. The server predicts the binding free energy change, the changes of the corresponding energy components and provides the energy minimized 3D structure of the wild type and mutant proteins for download. The SAAMBE protocol performance was tested by benchmarking the predictions against over 1300 experimentally determined changes of binding free energy and a Pearson correlation coefficient of 0.62 was obtained. How the predictions can be used for discriminating disease-causing from harmless mutations is discussed. The webserver can be accessed via http://compbio.clemson.edu/saambe_webserver/.

  19. Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity

    Directory of Open Access Journals (Sweden)

    Drew Michael GB

    2006-03-01

    Full Text Available Abstract Background MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions. Results A large dataset comprising MHC-peptide structural complexes was created by re-modelling pre-determined x-ray crystallographic structures. Static energetic analysis, following energy minimisation, was performed on the dataset in order to characterise interactions between bound peptides and the MHC Class I molecule, partitioning the interactions within the groove into van der Waals, electrostatic and total non-bonded energy contributions. Conclusion The QSAR techniques of Genetic Function Approximation (GFA and Genetic Partial Least Squares (G/PLS algorithms were used to identify key interactions between the two molecules by comparing the calculated energy values with experimentally-determined BL50 data. Although the peptide termini binding interactions help ensure the stability of the MHC Class I-peptide complex, the central region of the peptide is also important in defining the specificity of the interaction. As thermodynamic studies indicate that peptide association and dissociation may be driven entropically, it may be necessary to incorporate entropic contributions into future calculations.

  20. Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain.

    Science.gov (United States)

    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

  1. A New Method for Navigating Optimal Direction for Pulling Ligand from Binding Pocket: Application to Ranking Binding Affinity by Steered Molecular Dynamics.

    Science.gov (United States)

    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

  2. Curcumin binds in silico to anti-cancer drug target enzyme MMP-3 (human stromelysin-1) with affinity comparable to two known inhibitors of the enzyme.

    Science.gov (United States)

    Jerah, Ahmed; Hobani, Yahya; Kumar, B Vinod; Bidwai, Anil

    2015-01-01

    In silico interaction of curcumin with the enzyme MMP-3 (human stromelysin-1) was studied by molecular docking using AutoDock 4.2 as the docking software application. AutoDock 4.2 software serves as a valid and acceptable docking application to study the interactions of small compounds with proteins. Interactions of curcumin with MMP-3 were compared to those of two known inhibitors of the enzyme, PBSA and MPPT. The calculated free energy of binding (ΔG binding) shows that curcumin binds with affinity comparable to or better than the two known inhibitors. Binding interactions of curcumin with active site residues of the enzyme are also predicted. Curcumin appears to bind in an extendended conformation making extensive VDW contacts in the active site of the enzyme. Hydrogen bonding and pi-pi interactions with key active site residues is also observed. Thus, curcumin can be considered as a good lead compound in the development of new inhibitors of MMP-3 which is a potential target of anticancer drugs. The results of these studies can serve as a starting point for further computational and experimental studies. PMID:26420919

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

    OpenAIRE

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

    2001-01-01

    Abstract 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...

  4. NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Lamberth, K; Harndahl, M;

    2008-01-01

    NetMHC-3.0 is trained on a large number of quantitative peptide data using both affinity data from the Immune Epitope Database and Analysis Resource (IEDB) and elution data from SYFPEITHI. The method generates high-accuracy predictions of major histocompatibility complex (MHC): peptide binding. The...... predictions are based on artificial neural networks trained on data from 55 MHC alleles (43 Human and 12 non-human), and position-specific scoring matrices (PSSMs) for additional 67 HLA alleles. As only the MHC class I prediction server is available, predictions are possible for peptides of length 8–11 for...... all 122 alleles. artificial neural network predictions are given as actual IC50 values whereas PSSM predictions are given as a log-odds likelihood scores. The output is optionally available as download for easy post-processing. The training method underlying the server is the best available, and has...

  5. A community resource benchmarking predictions of peptide binding to MHC-I molecules.

    Directory of Open Access Journals (Sweden)

    Bjoern Peters

    2006-06-01

    Full Text Available Recognition of peptides bound to major histocompatibility complex (MHC class I molecules by T lymphocytes is an essential part of immune surveillance. Each MHC allele has a characteristic peptide binding preference, which can be captured in prediction algorithms, allowing for the rapid scan of entire pathogen proteomes for peptide likely to bind MHC. Here we make public a large set of 48,828 quantitative peptide-binding affinity measurements relating to 48 different mouse, human, macaque, and chimpanzee MHC class I alleles. We use this data to establish a set of benchmark predictions with one neural network method and two matrix-based prediction methods extensively utilized in our groups. In general, the neural network outperforms the matrix-based predictions mainly due to its ability to generalize even on a small amount of data. We also retrieved predictions from tools publicly available on the internet. While differences in the data used to generate these predictions hamper direct comparisons, we do conclude that tools based on combinatorial peptide libraries perform remarkably well. The transparent prediction evaluation on this dataset provides tool developers with a benchmark for comparison of newly developed prediction methods. In addition, to generate and evaluate our own prediction methods, we have established an easily extensible web-based prediction framework that allows automated side-by-side comparisons of prediction methods implemented by experts. This is an advance over the current practice of tool developers having to generate reference predictions themselves, which can lead to underestimating the performance of prediction methods they are not as familiar with as their own. The overall goal of this effort is to provide a transparent prediction evaluation allowing bioinformaticians to identify promising features of prediction methods and providing guidance to immunologists regarding the reliability of prediction tools.

  6. Structure-based rational design of a Toll-like receptor 4 (TLR4 decoy receptor with high binding affinity for a target protein.

    Directory of Open Access Journals (Sweden)

    Jieun Han

    Full Text Available Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4 decoy receptor composed of leucine-rich repeat (LRR modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2. Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (K(D one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities.

  7. Rapid and accurate prediction and scoring of water molecules in protein binding sites.

    Directory of Open Access Journals (Sweden)

    Gregory A Ross

    Full Text Available Water plays a critical role in ligand-protein interactions. However, it is still challenging to predict accurately not only where water molecules prefer to bind, but also which of those water molecules might be displaceable. The latter is often seen as a route to optimizing affinity of potential drug candidates. Using a protocol we call WaterDock, we show that the freely available AutoDock Vina tool can be used to predict accurately the binding sites of water molecules. WaterDock was validated using data from X-ray crystallography, neutron diffraction and molecular dynamics simulations and correctly predicted 97% of the water molecules in the test set. In addition, we combined data-mining, heuristic and machine learning techniques to develop probabilistic water molecule classifiers. When applied to WaterDock predictions in the Astex Diverse Set of protein ligand complexes, we could identify whether a water molecule was conserved or displaced to an accuracy of 75%. A second model predicted whether water molecules were displaced by polar groups or by non-polar groups to an accuracy of 80%. These results should prove useful for anyone wishing to undertake rational design of new compounds where the displacement of water molecules is being considered as a route to improved affinity.

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

    International Nuclear Information System (INIS)

    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 125I-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-125I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein

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

    Energy Technology Data Exchange (ETDEWEB)

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F. (Univ. of Tokyo (Japan))

    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.

  10. Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

    Science.gov (United States)

    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. PMID:25061691

  11. Direct Labeling of Polyphosphate at the Ultrastructural Level in Saccharomyces cerevisiae by Using the Affinity of the Polyphosphate Binding Domain of Escherichia coli Exopolyphosphatase

    OpenAIRE

    Saito, Katsuharu; Ohtomo, Ryo; Kuga-Uetake, Yukari; Aono, Toshihiro; Saito, Masanori

    2005-01-01

    Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affi...

  12. Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary

    International Nuclear Information System (INIS)

    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 125I-labeled human choriogonadotropin, with an equilibrium dissociation constant (Kd) of 2.5 x 10-9 M, which is comparable to the Kd 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 Mr 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 Mr 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the Mr 135,000 species was almost completely converted to a Mr 92,000 band, but the Mr 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 Mr 92,000 subunit, which comprises the hormone-binding domain

  13. Correlation and prediction of adsorption capacity and affinity of aromatic compounds on carbon nanotubes.

    Science.gov (United States)

    Wu, Wenhao; Yang, Kun; Chen, Wei; Wang, Wendi; Zhang, Jie; Lin, Daohui; Xing, Baoshan

    2016-01-01

    Adsorption of 22 nonpolar and polar aromatic compounds on 10 carbon nanotubes (CNTs) with various diameters, lengths and surface oxygen-containing group contents was investigated to develop predictive correlations for adsorption, using the isotherm fitting of Polanyi theory-based Dubinin-Ashtakhov (DA) model. Adsorption capacity of aromatic compounds on CNTs is negatively correlated with melting points of aromatic compounds, and surface oxygen-containing group contents and surface area ratios of mesopores to total pores of CNTs, but positively correlated with total surface area of CNTs. Adsorption affinity is positively correlated with solvatochromic parameters of aromatic compounds, independent of tube lengths and surface oxygen-containing group contents of CNTs, but negatively correlated with surface area ratios of mesopores to total pores of CNTs. The correlations of adsorption capacity and adsorption affinity with properties of both aromatic compounds and CNTs clearly have physical significance, can be used successfully with DA model to predict adsorption of aromatic compounds on CNTs from the well-known physiochemical properties of aromatic compounds (i.e., solvatochromic parameters, melting points) and CNTs (i.e., surface area and total acidic group contents), and thus can facilitate the environmental application of CNTs as sorbents and environmental risk assessment of both aromatic contaminants and CNTs. PMID:26521219

  14. ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

    Directory of Open Access Journals (Sweden)

    Anuradha Kuchibhatla

    Full Text Available A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0 pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

  15. The Structure of the Amyloid-β Peptide High-Affinity Copper II Binding Site in Alzheimer Disease

    International Nuclear Information System (INIS)

    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-β (Aβ) protein bound primarily to copper ions. The evidence for an oxidative stress role of Aβ-Cu redox chemistry is still incomplete. Details of the copper binding site in Aβ 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 Aβ peptides complexed with Cu2+ 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 Aβ-Cu2+ peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the Aβ-Cu2+ complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu2+ binding site is consistent with the hypothesis that the redox activity of the metal ion bound to Aβ can lead to the formation of dityrosine-linked dimers found in AD.

  16. Does antibody binding to diverse antigens predict future infection?

    Science.gov (United States)

    Owen, J P; Waite, J L; Holden, K Z; Clayton, D H

    2014-11-01

    We studied diverse antigen binding in hosts and the outcome of parasitism. We used captive-bred F1 descendants of feral rock pigeons (Columba livia) challenged with blood-feeding flies (Hippoboscidae) and a protozoan parasite (Haemoproteus). Enzyme-linked immunosorbent assays (ELISAs) and immunoblots were used to test (i) whether pre-infection IgY antigen binding predicts parasite fitness and (ii) whether antigen binding changes after infection. Assays used extracts from three pigeon parasites (northern fowl mite, Salmonella bacteria and avian pox virus), as well as nonparasitic molecules from cattle, chicken and keyhole limpet. Binding to hippoboscid and S. enterica extracts were predictive of hippoboscid fly fitness. Binding to extracts from hippoboscids, pox virus and nonparasitic organisms was predictive of Haemoproteus infection levels. Antigen binding to all extracts increased after parasite challenge, despite the fact that birds were only exposed to flies and Haemoproteus. Immunoblots suggested innate Ig binding to parasite-associated molecular markers and revealed that new antigens were bound in extracts after infection. These data suggest that host antibody binding to diverse antigens predicts parasite fitness even when the antigens are not related to the infecting parasite. We discuss the implications of these data for the study of host-parasite immunological interaction. PMID:25313676

  17. GHB receptor targets in the CNS: Focus on high-affinity binding sites

    DEFF Research Database (Denmark)

    Bay, Tina; Eghorn, Laura Friis; Klein, Anders Bue;

    2014-01-01

    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...

  18. A combined prediction strategy increases identification of peptides bound with high affinity and stability to porcine MHC class I molecules SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Rasmussen, Michael; Harndahl, Mikkel;

    2016-01-01

    constitute an attractive protocol to select target peptides from the vast pool of viral proteome peptides. We have earlier reported the peptide binding motif of the porcine MHC-I molecules SLA-1*04:01 and SLA-2*04:01, identified by an ELISA affinity-based positional scanning combinatorial peptide library...... (PSCPL) approach. Here, we report the peptide binding motif of SLA-3*04:01 and combine two prediction methods and analysis of both peptide binding affinity and stability of peptide-MHC complexes to improve rational peptide selection. Using a peptide prediction strategy combining PSCPL binding matrices...... and in silico prediction algorithms (NetMHCpan), peptide ligands from a repository of 8900 peptides were predicted for binding to SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01 and validated by affinity and stability assays. From the pool of predicted peptides for SLA-1*04:01, SLA-2*04:01, and SLA-3...

  19. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    Science.gov (United States)

    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

  20. Insights into affinity and specificity in the complexes of α-lytic protease and its inhibitor proteins: binding free energy from molecular dynamics simulation†

    OpenAIRE

    Deng, Nan-Jie; Cieplak, Piotr

    2009-01-01

    We report the binding free energy calculation and its decomposition for the complexes of α-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have va...

  1. Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD

    OpenAIRE

    Andrade, Fábia K; Moreira, Susana Margarida Gomes; Domingues, Lucília; Gama, F. M.

    2010-01-01

    The attachment of cells to biomedical materials can be improved by using adhesion sequences, such as Arg-Gly-Asp (RGD), found in several extracellular matrix proteins. In this work, bifunctional recombinant proteins, with a Cellulose-Binding Module (CBM), from the cellulosome of Clostridium thermocellum and cell binding sequences - RGD, GRGDY - were cloned and expressed in E.coli. These RGD-containing cellulose binding proteins were purified and used to coat bacterial cellulose fibres. Its ef...

  2. Cloud computing approaches for prediction of ligand binding poses and pathways.

    Science.gov (United States)

    Lawrenz, Morgan; Shukla, Diwakar; Pande, Vijay S

    2015-01-01

    We describe an innovative protocol for ab initio prediction of ligand crystallographic binding poses and highly effective analysis of large datasets generated for protein-ligand dynamics. We include a procedure for setup and performance of distributed molecular dynamics simulations on cloud computing architectures, a model for efficient analysis of simulation data, and a metric for evaluation of model convergence. We give accurate binding pose predictions for five ligands ranging in affinity from 7 nM to > 200 μM for the immunophilin protein FKBP12, for expedited results in cases where experimental structures are difficult to produce. Our approach goes beyond single, low energy ligand poses to give quantitative kinetic information that can inform protein engineering and ligand design. PMID:25608737

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

    Science.gov (United States)

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

    2012-05-01

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

  4. Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism.

    Science.gov (United States)

    Glaring, Mikkel A; Baumann, Martin J; Abou Hachem, Maher; Nakai, Hiroyuki; Nakai, Natsuko; Santelia, Diana; Sigurskjold, Bent W; Zeeman, Samuel C; Blennow, Andreas; Svensson, Birte

    2011-04-01

    Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism. PMID:21294843

  5. Influence of binding energies of electrons on nuclear mass predictions

    Science.gov (United States)

    Tang, Jing; Niu, Zhong-Ming; Guo, Jian-You

    2016-07-01

    Nuclear mass contains a wealth of nuclear structure information, and has been widely employed to extract the nuclear effective interactions. The known nuclear mass is usually extracted from the experimental atomic mass by subtracting the masses of electrons and adding the binding energy of electrons in the atom. However, the binding energies of electrons are sometimes neglected in extracting the known nuclear masses. The influence of binding energies of electrons on nuclear mass predictions are carefully investigated in this work. If the binding energies of electrons are directly subtracted from the theoretical mass predictions, the rms deviations of nuclear mass predictions with respect to the known data are increased by about 200 keV for nuclei with Z, N ⩾ 8. Furthermore, by using the Coulomb energies between protons to absorb the binding energies of electrons, their influence on the rms deviations is significantly reduced to only about 10 keV for nuclei with Z, N ⩾ 8. However, the binding energies of electrons are still important for the heavy nuclei, about 150 keV for nuclei around Z = 100 and up to about 500 keV for nuclei around Z = 120. Therefore, it is necessary to consider the binding energies of electrons to reliably predict the masses of heavy nuclei at an accuracy of hundreds of keV. Supported by National Natural Science Foundation of China (11205004)

  6. Changes in parathyroid hormone receptor binding affinity during egg laying: implications for calcium homeostasis in chicken.

    Science.gov (United States)

    Yasuoka, T; Kawashima, M; Takahashi, T; Iwata, A; Oka, N; Tanaka, K

    1996-12-01

    Parathyroid hormone (PTH) receptor bindings were examined in the membrane fraction of the calvaria and the kidney of the hen by the use of [125I]PTH-related protein (PTHrP) binding assays. The binding specificity, reversibility, and saturation of the receptor were demonstrated. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) were obtained by Scatchard analyses. In both calvaria and kidney, Kd and Bmax values decreased at 3 h before oviposition in egg-laying hens, but not in nonlaying hens. Administration of 17 beta-estradiol or progesterone in vivo caused a decrease in the Kd and Bmax values. Ionized calcium concentrations in the blood plasma showed a decrease at 13 h before oviposition. The results suggest that the PTH receptor binding in the calvaria and the kidney is affected by ovarian steroid hormones and may play a role in maintaining the calcium homeostasis in the egg-laying hen. PMID:8970893

  7. Prediction of the sorption capacities and affinities of organic chemicals by XAD-7.

    Science.gov (United States)

    Yang, Kun; Qi, Long; Wei, Wei; Wu, Wenhao; Lin, Daohui

    2016-01-01

    Macro-porous resins are widely used as adsorbents for the treatment of organic contaminants in wastewater and for the pre-concentration of organic solutes from water. However, the sorption mechanisms for organic contaminants on such adsorbents have not been systematically investigated so far. Therefore, in this study, the sorption capacities and affinities of 24 organic chemicals by XAD-7 were investigated and the experimentally obtained sorption isotherms were fitted to the Dubinin-Ashtakhov model. Linear positive correlations were observed between the sorption capacities and the solubilities (SW) of the chemicals in water or octanol and between the sorption affinities and the solvatochromic parameters of the chemicals, indicating that the sorption of various organic compounds by XAD-7 occurred by non-linear partitioning into XAD-7, rather than by adsorption on XAD-7 surfaces. Both specific interactions (i.e., hydrogen-bonding interactions) as well as nonspecific interactions were considered to be responsible for the non-linear partitioning. The correlation equations obtained in this study allow the prediction of non-linear partitioning using well-known chemical parameters, namely SW, octanol-water partition coefficients (KOW), and the hydrogen-bonding donor parameter (αm). The effect of pH on the sorption of ionizable organic compounds (IOCs) could also be predicted by combining the correlation equations with additional equations developed from the estimation of IOC dissociation rates. The prediction equations developed in this study and the proposed non-linear partition mechanism shed new light on the selective removal and pre-concentration of organic solutes from water and on the regeneration of exhausted XAD-7 using solvent extraction. PMID:25561259

  8. Moringa oleifera lectin and binding affinity to humic acids : application to water treatment

    OpenAIRE

    Santos, Andréa F. S.; Luz, Luciana A.; Cunha, M. G. C.; Paiva, Patrícia M.G.; Teixeira, J.A.; Brito, A. G.; R. Nogueira; L. C. B. B. Coelho

    2007-01-01

    Humic acids, natural organic matter, constitute a problem in water treatment; they react with chlorine forming undesirable disinfection byproducts. The aim of this work was the purification of a saline soluble Moringa oleifera lectin (SSMoL) and characterization of its affinity to humic acids. SSMoL isolation included extraction of seed flour, saline fractionation and guar gel chromatography. Hemagglutinating activity (HA) of extract (E), fraction (0-60F) and SSMoL was assessed...

  9. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation...... occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions....... Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in...

  10. Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts

    DEFF Research Database (Denmark)

    Woods, A; Longley, R L; Tumova, S;

    2000-01-01

    fibroblasts attach and spread following integrin ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component......Cell adhesion to extracellular matrix involves signaling mechanisms which control attachment, spreading and the formation of focal adhesions and stress fibers. Fibronectin can provide sufficient signals for all three processes, even when protein synthesis is prevented by cycloheximide. Primary...... present together with integrins in focal adhesions. Syndecan-4 binds and activates protein kinase Calpha, whose activity is needed for focal adhesion formation. We now report that the glycosaminoglycan chains of syndecan-4 bind recombinant HepII and it is incorporated into forming focal adhesions....

  11. Influence of the high-affinity growth hormone (GH)-binding protein on plasma profiles of free and bound GH and on the apparent half-life of GH. Modeling analysis and clinical applications.

    OpenAIRE

    Veldhuis, J D; Johnson, M.L.; Faunt, L M; MERCADO, M.; Baumann, G

    1993-01-01

    The discovery of a specific high-affinity growth hormone (GH) binding protein (GH-BP) in plasma adds complexity to the dynamics of GH secretion and clearance. Intuitive predictions are that such a protein would damp sharp oscillations in GH concentrations otherwise caused by bursts of GH secretion into the blood volume, prolong the apparent half-life of circulating GH, and contribute a reservoir function. To test these implicit considerations, we formulated an explicit mathematical model of p...

  12. Starch‐binding domains in the CBM45 family – low‐affinity domains from glucan, water dikinase and α‐amylase involved in plastidial starch metabolism

    DEFF Research Database (Denmark)

    Glaring, Mikkel Andreas; Baumann, Martin; Abou Hachem, Maher;

    2011-01-01

    organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45‐type domains, the Solanum tuberosumα‐glucan, water dikinase and the Arabidopsis thaliana plastidial α‐amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry......Starch‐binding domains are noncatalytic carbohydrate‐binding modules that mediate binding to granular starch. The starch‐binding domains from the carbohydrate‐binding module family 45 (CBM45, ) are found as N‐terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing...... extracellular microbial amylolytic enzymes. This suggests that low‐affinity starch‐binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low‐affinity interaction with starch granules, facilitates dynamic regulation of...

  13. Gestational treatment with cocaine and fluoxetine alters oxytocin receptor number and binding affinity in lactating rat dams.

    Science.gov (United States)

    Johns, Josephine M; Lubin, Deborah A; Walker, Cheryl H; Joyner, Paul; Middleton, Christopher; Hofler, Vivian; McMurray, Matthew

    2004-01-01

    Cocaine administered chronically throughout gestation has been correlated with deficits in maternal behavior, increased maternal aggressive behavior and decreased oxytocin levels in rats. In addition to its effects on oxytocin levels, cocaine is a potent serotonergic, dopaminergic and noradrenergic reuptake inhibitor. Alterations in the dopaminergic and serotonergic systems have been suggested as possibly having a role in cocaine-induced maternal aggression. This study was in part, an attempt to understand some of the mechanisms by which cocaine increases postpartum aggression, particularly as they relate to changes in the oxytocin system. Oxytocin receptor number and binding affinity in the medial preoptic area of the hypothalamus, ventral tegmental area, hippocampus and amygdala were determined for lactating rat dams on postpartum day 6 (PPD 6) that were gestationally treated with cocaine, fluoxetine, saline or an amfonelic acid/fluoxetine drug combination. Cocaine and fluoxetine treatment both resulted in a significant up-regulation of oxytocin receptor number and lower receptor affinity in the amygdala of lactating rat dams compared to saline controls and the amfonelic acid/fluoxetine combination treatment group. Cocaine treatment also resulted in a significant down-regulation of oxytocin receptors in the medial preoptic area and both cocaine and fluoxetine treated dams had the highest affinity for oxytocin receptors in this brain region. Results of the present study support previous data indicating that alterations in oxytocinergic and perhaps serotonergic system dynamics in the amygdala may play a role in cocaine-induced postpartum aggression. PMID:15380831

  14. Polymerization Degrees, Molecular Weights and Protein-Binding Affinities of Condensed Tannin Fractions from a Leucaena leucocephala Hybrid

    Directory of Open Access Journals (Sweden)

    Mookiah Saminathan

    2014-06-01

    Full Text Available Condensed tannins (CTs form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1–F5 measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight—from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

  15. DNA-binding residues and binding mode prediction with binding-mechanism concerned models

    OpenAIRE

    Oyang Yen-Jen; Liu Yu-Cheng; Huang Chun-Chin; Huang Yu-Feng; Huang Chien-Kang

    2009-01-01

    Abstract Background Protein-DNA interactions are essential for fundamental biological activities including DNA transcription, replication, packaging, repair and rearrangement. Proteins interacting with DNA can be classified into two categories of binding mechanisms - sequence-specific and non-specific binding. Protein-DNA specific binding provides a mechanism to recognize correct nucleotide base pairs for sequence-specific identification. Protein-DNA non-specific binding shows sequence indepe...

  16. Involvement of nitrogen functional groups in high-affinity copper binding in tomato and wheat root apoplasts: spectroscopic and thermodynamic evidence.

    Science.gov (United States)

    Guigues, Stéphanie; Bravin, Matthieu N; Garnier, Cédric; Masion, Armand; Chevassus-Rosset, Claire; Cazevieille, Patrick; Doelsch, Emmanuel

    2016-03-16

    Carboxylic groups located in plant cell walls (CW) are generally considered to be the main copper binding sites in plant roots, despite the presence of other functional groups. The aim of this study was to investigate sites responsible for copper binding in root apoplasts, i.e. CW and outer surface of the plasma membrane (PM) continuum. Binding sites in root apoplasts were investigated by comparing isolated CW of a monocotyledon (Triticum aestivum L.) and dicotyledon (Solanum lycopersicum L.) crop with their respective whole roots. Copper speciation was examined by X-ray absorption (XAS) and (13)C-nuclear magnetic resonance spectroscopies while the affinity of ligands involved in copper binding was investigated by modeling copper sorption isotherms. Homogeneous speciation and binding of copper was found in wheat and tomato root apoplasts. Only Cu-N and Cu-O bonds were detected in wheat and tomato root apoplasts. Nitrogen/oxygen ligands were identified in slightly higher proportions (40-70%) than single oxygen ligands. Furthermore, low- and high-affinity binding sites contributed in an almost equivalent proportion to copper binding in root apoplasts. The high-affinity N functional groups embedded in root apoplasts participated in copper binding in the same magnitude than the low-affinity carboxylic groups. PMID:26824877

  17. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.; Kramhoft, B.; Svensson, Birte

    2006-01-01

    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 b...

  18. The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin TransporterⓈ

    OpenAIRE

    Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H; Ecker, Gerhard F.; Freissmuth, Michael; Sucic, Sonja

    2010-01-01

    The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homolo...

  19. DETERMINANTS OF LIGAND BINDING AFFINITY AND COOPERATIVITY AT THE GLUT1 ENDOFACIAL SITE

    OpenAIRE

    Robichaud, Trista; Appleyard, Antony N.; Herbert, Richard B.; Henderson, Peter J. F.; Carruthers, Anthony

    2011-01-01

    Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport (Cloherty, E. K., Levine, K. B., & Carruthers, A. (2001). The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry, 40(51), 15549–15561). This result is consistent with t...

  20. Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.

    Science.gov (United States)

    Ozoe, Yoshihisa; Kita, Tomo; Ozoe, Fumiyo; Nakao, Toshifumi; Sato, Kazuyuki; Hirase, Kangetsu

    2013-11-01

    γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides

  1. Characterization of valinomycin binding with alkali metal ions by capillary affinity electrophoresis and density functional theory

    Czech Academy of Sciences Publication Activity Database

    Ehala, Sille; Dybal, Jiří; Makrlík, E.; Kašička, Václav

    Praha : Institut of organic Chemistry and Biochemistry ASCR, 2009 - (Slaninová, J.), s. 31-33 ISBN 978-80-86241-31-9. - (Collection Symposium Series. 11). [Biologically Active Peptides. Conference /11./. Praha (CZ), 22.04.2009-24.04.2009] R&D Projects: GA ČR(CZ) GA203/08/1428; GA AV ČR 1ET400500402 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z40500505 Keywords : capillary affinity electrophoresis * valinomycin * density funtional theory Subject RIV: CF - Physical ; Theoretical Chemistry

  2. Artifactual high-affinity and saturable binding of (3H)5-hydroxytryptamine induced by radioligand oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Peroutka, S.J.; Ison, P.J.; Liu, D.U.; Barrett, R.W.

    1986-07-01

    The binding of (3H)5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of (/sup 3/H)5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM (/sup 3/H)5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of (/sup 3/H)5-HT in the absence of ascorbate. The increase in apparent specific (/sup 3/H)5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of (/sup 3/H)5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific (/sup 3/H)5-HT binding in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that (3H)5-HT is essentially destroyed by a 4-h incubation at 22/sup 0/C in the absence of ascorbate.

  3. The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

    Science.gov (United States)

    Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

    2015-01-01

    The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates. PMID:25658443

  4. Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.

    Science.gov (United States)

    Chauvaux, S; Beguin, P; Aubert, J P; Bhat, K M; Gow, L A; Wood, T M; Bairoch, A

    1990-01-01

    Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region. Images Fig. 1. Fig. 3. PMID:2302168

  5. Prediction of MHC class I binding peptides, using SVMHC

    Directory of Open Access Journals (Sweden)

    Elofsson Arne

    2002-09-01

    Full Text Available Abstract Background T-cells are key players in regulating a specific immune response. Activation of cytotoxic T-cells requires recognition of specific peptides bound to Major Histocompatibility Complex (MHC class I molecules. MHC-peptide complexes are potential tools for diagnosis and treatment of pathogens and cancer, as well as for the development of peptide vaccines. Only one in 100 to 200 potential binders actually binds to a certain MHC molecule, therefore a good prediction method for MHC class I binding peptides can reduce the number of candidate binders that need to be synthesized and tested. Results Here, we present a novel approach, SVMHC, based on support vector machines to predict the binding of peptides to MHC class I molecules. This method seems to perform slightly better than two profile based methods, SYFPEITHI and HLA_BIND. The implementation of SVMHC is quite simple and does not involve any manual steps, therefore as more data become available it is trivial to provide prediction for more MHC types. SVMHC currently contains prediction for 26 MHC class I types from the MHCPEP database or alternatively 6 MHC class I types from the higher quality SYFPEITHI database. The prediction models for these MHC types are implemented in a public web service available at http://www.sbc.su.se/svmhc/. Conclusions Prediction of MHC class I binding peptides using Support Vector Machines, shows high performance and is easy to apply to a large number of MHC class I types. As more peptide data are put into MHC databases, SVMHC can easily be updated to give prediction for additional MHC class I types. We suggest that the number of binding peptides needed for SVM training is at least 20 sequences.

  6. High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

    Science.gov (United States)

    Ullman, Christopher; Mathonet, Pascale; Oleksy, Arkadiusz; Diamandakis, Agata; Tomei, Licia; Demartis, Anna; Nardi, Chiara; Sambucini, Sonia; Missineo, Antonino; Alt, Karen; Hagemeyer, Christoph E.; Harris, Matt; Hedt, Amos; Weis, Roland; Gehlsen, Kurt R.

    2015-01-01

    Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model. PMID:26313909

  7. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Directory of Open Access Journals (Sweden)

    Jonathan S Wall

    Full Text Available Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  8. Reliable prediction of transcription factor binding sites by phylogenetic verification

    OpenAIRE

    Li, Xiaoman; Zhong, Sheng; Wong, Wing H.

    2005-01-01

    We present a statistical methodology that largely improves the accuracy in computational predictions of transcription factor (TF) binding sites in eukaryote genomes. This method models the cross-species conservation of binding sites without relying on accurate sequence alignment. It can be coupled with any motif-finding algorithm that searches for overrepresented sequence motifs in individual species and can increase the accuracy of the coupled motif-finding algorithm. Because this method is ...

  9. Affinity tagging & purification of the fucose binding LecB protein

    OpenAIRE

    Creavin, Aileen; O'Connor, Brendan

    2007-01-01

    The fucose binding LecB protein is one of two identified lectins produced by the opportunistic pathogen Pseudomonas aeruginosa (PA01) and is implicated in contributing to its virulence. A large number of homologous proteins have been identified in other bacterial species that exhibit extremely high sequence identity and similarity to LecB. However, key amino acid residues known to participate in fucose binding in LecB are altered in many of these proteins. Some of these proteins have been sho...

  10. Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus embryos

    OpenAIRE

    Bogdanović Ozren; Veenstra Gert Jan C

    2011-01-01

    Abstract Background DNA methylation is a widespread epigenetic modification in vertebrate genomes. Genomic sites of DNA methylation can be bound by methyl-CpG-binding domain proteins (MBDs) and specific zinc finger proteins, which can recruit co-repressor complexes to silence transcription on targeted loci. The binding to methylated DNA may be regulated by post-translational MBD modifications. Findings A methylated DNA affinity precipitation method was implemented to assay binding of proteins...

  11. 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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

    Science.gov (United States)

    Cubrilovic, Dragana; Biela, Adam; Sielaff, Frank; Steinmetzer, Torsten; Klebe, Gerhard; Zenobi, Renato

    2012-10-01

    NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solution.

  13. Predicting Binding Free Energy Change Caused by Point Mutations with Knowledge-Modified MM/PBSA Method

    OpenAIRE

    Marharyta Petukh; Minghui Li; Emil Alexov

    2015-01-01

    Author Summary Developing methods for accurate prediction of effects of amino acid substitutions on protein-protein affinity is important for both understanding disease-causing mechanism of missense mutations and guiding protein engineering. For both purposes, there is a need for accurate methods primarily based on first principle calculations, while being fast enough to handle large number of cases. Here we report a new method, the Single Amino Acid Mutation based change in Binding free Ener...

  14. A systems biology approach to transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    Full Text Available BACKGROUND: The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs, identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates. METHODOLOGY/PRINCIPAL FINDINGS: We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data. CONCLUSIONS/SIGNIFICANCE: Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct

  15. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination

    DEFF Research Database (Denmark)

    Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan; Jensen, Lars Juhl; Berthelsen, Jens

    2014-01-01

    Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening...

  16. Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein

    Czech Academy of Sciences Publication Activity Database

    Uhlík, Ondřej; Kamlar, Marek; Kohout, Ladislav; Ježek, Rudolf; Harmatha, Juraj; Macek, Tomáš

    2008-01-01

    Roč. 73, č. 14 (2008), s. 1433-1440. ISSN 0039-128X R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z40550506 Keywords : ecdysteroids * RuBisCO * regulation * binding protein Subject RIV: CC - Organic Chemistry Impact factor: 2.588, year: 2008

  17. Automated evaluation of protein binding affinity of anti-inflammatory choline based ionic liquids.

    Science.gov (United States)

    Ribeiro, Rosa; Pinto, Paula C A G; Azevedo, Ana M O; Bica, Katharina; Ressmann, Anna K; Reis, Salette; Saraiva, M Lúcia M F S

    2016-04-01

    In this work, an automated system for the study of the interaction of drugs with human serum albumin (HSA) was developed. The methodology was based on the quenching of the intrinsic fluorescence of HSA by binding of the drug to one of its binding sites. The fluorescence quenching assay was implemented in a sequential injection analysis (SIA) system and the optimized assay was applied to ionic liquids based on the association of non-steroidal anti-inflammatory drugs with choline (IL-API). In each cycle, 100µL of HSA and 100µL of IL-API (variable concentration) were aspirated at a flow rate of 1mLmin(-1) and then sent through the reaction coil to the detector where the fluorescence intensity was measured. In the optimized conditions the effect of increasing concentrations of choline ketoprofenate and choline naproxenate (and respective starting materials: ketoprofen and naproxen) on the intrinsic fluorescence of HSA was studied and the dissociation constants (Kd) were calculated by means of models of drug-protein binding in the equilibrium. The calculated Kd showed that all the compounds bind strongly to HSA (Kd<100µmolL(-1)) and that the use of the drugs in the IL format does not affect or can even improve their HSA binding. The obtained results were compared with those provided by a conventional batch assay and the relative errors were lower than 4.5%. The developed SIA methodology showed to be robust and exhibited good repeatability in all the assay conditions (rsd<6.5%). PMID:26838377

  18. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    Science.gov (United States)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  19. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry.

    Science.gov (United States)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects. Graphical Abstract ᅟ. PMID:27112153

  20. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    Science.gov (United States)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-04-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  1. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1, Val1, Asn2, Gln3, His4, Ser8, His9, Glu12, Tyr15, Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3, Ala4] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15, Leu16] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln3, Ala4, Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

  2. Leucine zipper like structure in rice WRKY89 enhances its affinity for binding with W box elements

    Institute of Scientific and Technical Information of China (English)

    WANG Haihua; HAO Zhongna; XIE Ke; WU Kunlu; GUO Zejian

    2005-01-01

    WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of OsWRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.

  3. Analysis of glipizide binding to normal and glycated human serum albumin by high-performance affinity chromatography.

    Science.gov (United States)

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-07-01

    In diabetes, the elevated levels of glucose in the bloodstream can result in the nonenzymatic glycation of proteins such as human serum albumin (HSA). This type of modification has been shown to affect the interactions of some drugs with HSA, including several sulfonylurea drugs that are used to treat type II diabetes. This study used high-performance affinity chromatography (HPAC) to examine the interactions of glipizide (i.e., a second-generation sulfonylurea drug) with normal HSA or HSA that contained various levels of in vitro glycation. Frontal analysis indicated that glipizide was interacting with both normal and glycated HSA through two general groups of sites: a set of relatively strong interactions and a set of weaker interactions with average association equilibrium constants at pH 7.4 and 37 °C in the range of 2.4-6.0 × 10(5) and 1.7-3.7 × 10(4) M(-1), respectively. Zonal elution competition studies revealed that glipizide was interacting at both Sudlow sites I and II, which were estimated to have affinities of 3.2-3.9 × 10(5) and 1.1-1.4 × 10(4) M(-1). Allosteric effects were also noted to occur for this drug between the tamoxifen site and the binding of R-warfarin at Sudlow site I. Up to an 18% decrease in the affinity for glipizide was observed at Sudlow site I ongoing from normal HSA to glycated HSA, while up to a 27% increase was noted at Sudlow site II. This information should be useful in indicating how HPAC can be used to investigate other drugs that have complex interactions with proteins. These results should also be valuable in providing a better understanding of how glycation may affect drug-protein interactions and the serum transport of drugs such as glipizide during diabetes. PMID:25912461

  4. Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

    International Nuclear Information System (INIS)

    The synthetic peptide containing residues 43-49 of α-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 α-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 α-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of [125I]α-gliadin(43-49) than T-lymphocytes. The lymphocyte α-gliadin(43-49) receptor may play an important role in mediating the immunological response to α-gliadin. 16 references, 4 figures

  6. Development of affinity technology for isolating individual human chromosomes by third strand binding

    Energy Technology Data Exchange (ETDEWEB)

    Fresco, Jacques R.

    2003-06-01

    The overall goal was to explore whether nucleic acid third strands could be used to bind with very high specificity to specific targets within whole genomes. Towards this end conditions had to be found to keep erroneous binding to an absolute minimum. The goal to use third strands (linked to magnetic beads) to ''capture'' large particles such as plasmids, cosmids, and whole chromosomes from complex mixtures was partially met; their use to serve as cytogenetic probes of metaphase chromosomes and to deliver reactive reagents to unique target sites on chromosomes in vivo for the purpose of mutagenizing specific base pairs was fully met; and their use as cytogenetic probes of chromosomal DNA in sections of formalin-fixed, paraffin-embedded tissue has been met since the DOE support was terminated.

  7. Tissue-binding affinity of Proteus mirabilis fimbriae in the human urinary tract.

    OpenAIRE

    Sareneva, T; Holthöfer, H; Korhonen, T K

    1990-01-01

    Binding characteristics of the two major fimbrial hemagglutinin types of uropathogenic Proteus mirabilis were determined in frozen sections of human kidney and in exfoliated uroepithelial cells. P. mirabilis 3087, which expresses the MR/P fimbriae, adhered avidly to the tubular epithelial cells of the kidney and also to the epithelial cells of urinary sediment. No adhesion to glomerular or peritubular elements of the kidney was detected. Indirect immunogold silver staining also showed that th...

  8. Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding

    OpenAIRE

    Magalhães, Maria L. B.; Czekster, Clarissa Melo; Guan, Rong; Malashkevich, Vladimir N.; Almo, Steven C.; Levy, Matthew

    2011-01-01

    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 cont...

  9. Interactions of dopamine agonists with brain D1 receptors labeled by 3H-antagonists. Evidence for the presence of high and low affinity agonist-binding states

    International Nuclear Information System (INIS)

    The interactions of dopaminergic agonists and antagonists with 3H-antagonist labeled D1 dopamine receptors of rat striatum have been characterized. [3H]Flupentixol has been found to selectively label D1 dopamine receptors when its binding to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone. Antagonist/3H-antagonist competition curves are of uniformly steep slope (nH . 1.0) suggesting the presence of a single D1 dopamine receptor. Agonist/3H-antagonist competition curves are extremely shallow (nH less than or equal to 0.5) for agonists of high relative efficacy, suggesting the presence of heterogeneous populations of agonist-binding states of the D1 dopamine receptor. Computer-modeling techniques were used to estimate affinities and relative site densities for these heterogeneous binding states. This analysis indicates that the ratio of agonist affinities for low and high affinity agonist-binding states is correlated with agonist relative efficacies in activating adenylate cyclase in membrane homogenates. Under the assay conditions employed, the addition of saturating concentrations of guanine nucleotides reduced, but did not abolish, the relative density of high affinity agonist-binding sites. These binding data can, at least in part, be explained by postulating two states of the D1 dopamine receptor, inducible by agonists but not by antagonists and modulated by guanine nucleotides

  10. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Lina Lu

    2015-05-01

    Full Text Available Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed.

  11. An In-tether Chiral Center Modulates the Helicity, Cell Permeability, and Target Binding Affinity of a Peptide.

    Science.gov (United States)

    Hu, Kuan; Geng, Hao; Zhang, Qingzhou; Liu, Qisong; Xie, Mingsheng; Sun, Chengjie; Li, Wenjun; Lin, Huacan; Jiang, Fan; Wang, Tao; Wu, Yun-Dong; Li, Zigang

    2016-07-01

    The addition of a precisely positioned chiral center in the tether of a constrained peptide is reported, yielding two separable peptide diastereomers with significantly different helicity, as supported by circular dichroism (CD) and NMR spectroscopy. Single crystal X-ray diffraction analysis suggests that the absolute configuration of the in-tether chiral center in helical form is R, which is in agreement with theoretical simulations. The relationship between the secondary structure of the short peptides and their biochemical/biophysical properties remains elusive, largely because of the lack of proper controls. The present strategy provides the only method for investigating the influence of solely conformational differences upon the biochemical/biophysical properties of peptides. The significant differences in permeability and target binding affinity between the peptide diastereomers demonstrate the importance of helical conformation. PMID:27167181

  12. [3H]naloxone as an opioid receptor label: Analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues

    International Nuclear Information System (INIS)

    The nonselective antagonist [3H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opiod ligands as well as various β-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between μ1 and μ2 binding sites. The affinities to the μ sites obtained with [3H]naloxone as label are in a good agreement with those from experiments with the μ selective radioligand [3H]DAGO. The μ1 site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity μ1 subtype. (author)

  13. Kinetics of binding of dihydropyridine calcium channel ligands to skeletal muscle membranes: Evidence for low-affinity sites and for the involvement of G proteins

    International Nuclear Information System (INIS)

    Detailed kinetic studies of the binding of the calcium channel antagonist (+)-[3H]PN200-110 to membrane preparations form rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites that are readily measured in equilibrium and kinetic experiments, there are also dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-[3H]PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing ligand, and half-maximal effects occurred at approximately 10 μM for the agonist (±)-Bay K8644 and for the antagonists nifedipine, (±)-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereospecific since (-)-PN200-110 (1-200 μM) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-O-(3-thiotriphosphate) (GTPγS) and guanosine 5'-O-(2-thiodiphosphate) (GDPβS) on binding parameters. GTPγS did increase the ability of (±)-[3H]PN200-110. These results suggest that skeletal muscle dihydropyridine receptors have low-affinity binding sites that may be involved in the regulation of calcium channel function and that activation of a guanine nucleotide binding protein may modulate the binding of agonists but not of antagonists to these sites

  14. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction

    Directory of Open Access Journals (Sweden)

    Lund Ole

    2009-09-01

    Full Text Available Abstract Background The major histocompatibility complex (MHC molecule plays a central role in controlling the adaptive immune response to infections. MHC class I molecules present peptides derived from intracellular proteins to cytotoxic T cells, whereas MHC class II molecules stimulate cellular and humoral immunity through presentation of extracellularly derived peptides to helper T cells. Identification of which peptides will bind a given MHC molecule is thus of great importance for the understanding of host-pathogen interactions, and large efforts have been placed in developing algorithms capable of predicting this binding event. Results Here, we present a novel artificial neural network-based method, NN-align that allows for simultaneous identification of the MHC class II binding core and binding affinity. NN-align is trained using a novel training algorithm that allows for correction of bias in the training data due to redundant binding core representation. Incorporation of information about the residues flanking the peptide-binding core is shown to significantly improve the prediction accuracy. The method is evaluated on a large-scale benchmark consisting of six independent data sets covering 14 human MHC class II alleles, and is demonstrated to outperform other state-of-the-art MHC class II prediction methods. Conclusion The NN-align method is competitive with the state-of-the-art MHC class II peptide binding prediction algorithms. The method is publicly available at http://www.cbs.dtu.dk/services/NetMHCII-2.0.

  15. A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity

    OpenAIRE

    Scherf, Tali; Kasher, Roni; Balass, Moshe; Fridkin, Mati; Fuchs, Sara; Katchalski-Katzir, Ephraim

    2001-01-01

    Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure crea...

  16. The Affinity of the Dynein Microtubule-Binding Domain is Modulated by the Conformation of its Coiled-Coil Stalk*

    OpenAIRE

    Gibbons, I. R.; Garbarino, Joan E.; Tan, Carol E.; Reck-Peterson, Samara L; Vale, Ronald D.; Carter, Andrew P.

    2005-01-01

    The microtubule binding domain (MTBD) of dynein is separated from the AAA core of the motor by an ~15 nm stalk that is predicted to consist of an anti-parallel coiled coil. However, the structure of this coiled-coil and the mechanism it uses to mediate communication between the MTBD and ATP-binding core are unknown. Here, we sought to identify the optimal alignment between the hydrophobic heptad repeats in the two strands of the stalk coiled-coil. To do this, we fused the MTBD of mouse cytopl...

  17. Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi.

    Science.gov (United States)

    Meehan, M; Nowlan, P; Owen, P

    1998-04-01

    Cell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M(r) protein species (apparent M(r) 220,000 and 550,000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M(r) protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M(r) 54,597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M(r) estimation, fibrinogen binding and seroreactivity. PMID:9579073

  18. Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

    Science.gov (United States)

    Birchfield, N B; Casida, J E

    1996-01-01

    Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis. PMID:8902268

  19. High-affinity insulin binding to an atypical insulin-like growth factor-I receptor in human breast cancer cells.

    OpenAIRE

    Milazzo, G.; Yip, C. C.; Maddux, B A; Vigneri, R; Goldfine, I D

    1992-01-01

    We studied the nature of insulin receptor binding in MCF-7 breast cancer cells. In both intact cells and solubilized receptor preparations, high-affinity insulin binding was seen. However, unlabeled insulin-like growth factor-I (IGF-I) was five-fold more potent in inhibiting 125I-insulin binding than insulin itself. With monoclonal antibodies to the insulin receptor, 30% of 125I-insulin binding was inhibited. In contrast when alpha-IR3, a monoclonal antibody that recognizes typical IGF-I rece...

  20. Improved pan-specific MHC class I peptide-binding predictions using a novel representation of the MHC-binding cleft environment

    DEFF Research Database (Denmark)

    Carrasco Pro, S.; Zimic, M.; Nielsen, Morten

    2014-01-01

    Major histocompatibility complex (MHC) molecules play a key role in cell-mediated immune responses presenting bounded peptides for recognition by the immune system cells. Several in silico methods have been developed to predict the binding affinity of a given peptide to a specific MHC molecule. One...... made available, and also new structures of MHC class I molecules with a bound peptide have been published. In order to test if the NetMHCpan method can be improved by integrating this novel information, we created new pseudo-sequence definitions for the MHC-binding cleft environment from sequence and...... but also by a refined definition of the MHC-binding environment including information from non-human species....

  1. Reliable prediction of transcription factor binding sites by phylogenetic verification.

    Science.gov (United States)

    Li, Xiaoman; Zhong, Sheng; Wong, Wing H

    2005-11-22

    We present a statistical methodology that largely improves the accuracy in computational predictions of transcription factor (TF) binding sites in eukaryote genomes. This method models the cross-species conservation of binding sites without relying on accurate sequence alignment. It can be coupled with any motif-finding algorithm that searches for overrepresented sequence motifs in individual species and can increase the accuracy of the coupled motif-finding algorithm. Because this method is capable of accurately detecting TF binding sites, it also enhances our ability to predict the cis-regulatory modules. We applied this method on the published chromatin immunoprecipitation (ChIP)-chip data in Saccharomyces cerevisiae and found that its sensitivity and specificity are 9% and 14% higher than those of two recent methods. We also recovered almost all of the previously verified TF binding sites and made predictions on the cis-regulatory elements that govern the tight regulation of ribosomal protein genes in 13 eukaryote species (2 plants, 4 yeasts, 2 worms, 2 insects, and 3 mammals). These results give insights to the transcriptional regulation in eukaryotic organisms. PMID:16286651

  2. Radioiodinated ligands for the estrogen receptor: Effect of different 7-cyanoalkyl chains on the binding affinity of novel iodovinyl-6-dehydroestradiols

    International Nuclear Information System (INIS)

    Three novel 17α-ethynyl-Δ6,7-estra-3,17β-diols and their 17α-[125I]-iodovinyl derivatives, containing different C7-cyanoalkyl chains, were studied as potential radioligands for the estrogen receptor. The influence of the chain length on the biological behaviour of the compounds was assessed through in vitro ER binding assays of the ethynyl derivatives and breast cancer cell uptake studies of the 17α-[125I]-iodovinyl-Δ6,7-estra-3,17β-diols. A difference in alkyl chain induced a decrease in ER binding affinities of substances, however, the receptor-binding affinities (RBA) of all compounds were lower than that of estradiol itself. In addition, a non-specific cell binding was observed which is in accordance with the encountered ethynyl RBA values suggesting that the uptake is not ER mediated

  3. Radioiodinated ligands for the estrogen receptor: Effect of different 7-cyanoalkyl chains on the binding affinity of novel iodovinyl-6-dehydroestradiols

    Energy Technology Data Exchange (ETDEWEB)

    Neto, Carina [Instituto Tecnologico e Nuclear, Estrada Nacional 10, 2686-953 Sacavem (Portugal); Centro de Investigacao em Meio Ambiente Genetica e Oncobiologia (CIMAGO) (Portugal); Oliveira, Maria Cristina [Instituto Tecnologico e Nuclear, Estrada Nacional 10, 2686-953 Sacavem (Portugal); Centro de Investigacao em Meio Ambiente Genetica e Oncobiologia (CIMAGO) (Portugal)], E-mail: cmelo@itn.pt; Gano, Lurdes; Marques, Fernanda; Santos, Isabel [Instituto Tecnologico e Nuclear, Estrada Nacional 10, 2686-953 Sacavem (Portugal); Centro de Investigacao em Meio Ambiente Genetica e Oncobiologia (CIMAGO) (Portugal); Morais, Goreti Ribeiro [Faculty of Pharmacy, University of Lisbon, Lisbon (Portugal); Yasuda, Takumi [Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Fukuoka (Japan); Thiemann, Thies [Centro de Investigacao em Meio Ambiente Genetica e Oncobiologia (CIMAGO) (Portugal); Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Fukuoka (Japan); Botelho, Filomena [Centro de Investigacao em Meio Ambiente Genetica e Oncobiologia (CIMAGO) (Portugal); Instituto de Biofisica/Biomatematica, IBILI, FMUC, Coimbra (Portugal); Oliveira, Carlos F. [Centro de Investigacao em Meio Ambiente Genetica e Oncobiologia (CIMAGO) (Portugal); Clinica Ginecologica, FMUC, Coimbra (Portugal)

    2009-02-15

    Three novel 17{alpha}-ethynyl-{delta}{sup 6,7}-estra-3,17{beta}-diols and their 17{alpha}-[{sup 125}I]-iodovinyl derivatives, containing different C7-cyanoalkyl chains, were studied as potential radioligands for the estrogen receptor. The influence of the chain length on the biological behaviour of the compounds was assessed through in vitro ER binding assays of the ethynyl derivatives and breast cancer cell uptake studies of the 17{alpha}-[{sup 125}I]-iodovinyl-{delta}{sup 6,7}-estra-3,17{beta}-diols. A difference in alkyl chain induced a decrease in ER binding affinities of substances, however, the receptor-binding affinities (RBA) of all compounds were lower than that of estradiol itself. In addition, a non-specific cell binding was observed which is in accordance with the encountered ethynyl RBA values suggesting that the uptake is not ER mediated.

  4. (/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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Insights into affinity and specificity in the complexes of alpha-lytic protease and its inhibitor proteins: binding free energy from molecular dynamics simulation.

    Science.gov (United States)

    Deng, Nan-Jie; Cieplak, Piotr

    2009-07-01

    We report the binding free energy calculation and its decomposition for the complexes of alpha-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have vastly different affinity towards alpha-lytic protease (ALP), a bacterial serine protease. OMTKY3 inhibits the enzyme much more weakly (by approximately 10(6) times) than eglin C. Moreover, a variant of OMTKY3 with five mutations, OMTKY3M, has been shown to inhibit 10(4) times more strongly than the wild-type inhibitor. The underlying mechanisms for the unusually large difference in binding affinities and the effect of mutation are not well understood. Here we use molecular dynamics simulation with molecular mechanics-Poisson Boltzmann/surface area method (MM-PB/SA) to investigate quantitatively the binding specificity. The calculated absolute binding free energies correctly differentiate the thermodynamic stabilities of these protein complexes, but the magnitudes of the binding affinities are systematically overestimated. Analysis of the binding free energy components provides insights into the molecular mechanism of binding specificity. The large DeltaDeltaG(bind) between eglin C and wild type OMTKY3 towards ALP is mainly attributable to the stronger nonpolar interactions in the ALP-eglin C complex, arising from a higher degree of structural complementarity. Here the electrostatic interaction contributes to a lesser extent. The enhanced inhibition in the penta-mutant OMTKY3M over its wild type is entirely due to an overall improvement in the solvent-mediated electrostatic interactions in the ALP-OMTKY3M complex. The results suggest that for these protein-complexes and

  6. Dithiocarbamate/piperazine bridged pyrrolobenzodiazepines as DNA-minor groove binders: synthesis, DNA-binding affinity and cytotoxic activity.

    Science.gov (United States)

    Kamal, Ahmed; Sreekanth, Kokkonda; Shankaraiah, Nagula; Sathish, Manda; Nekkanti, Shalini; Srinivasulu, Vunnam

    2015-04-01

    A new series of C8-linked dithiocarbamate/piperazine bridged pyrrolo[2,1-c][1,4]benzodiazepine conjugates (5a-c, 6a,b) have been synthesized and evaluated for their cytotoxic potential and DNA-binding ability. The representative conjugates 5a and 5b have been screened for their cytotoxicity against a panel of 60 human cancer cell lines. Compound 5a has shown promising cytotoxic activity on selected cancer cell lines that display melanoma, leukemia, CNS, ovarian, breast and renal cancer phenotypes. The consequence of further replacement of the 3-cyano-3,3-diphenylpropyl 1-piperazinecarbodithioate in 5b and 5c with 4-methylpiperazine-1-carbodithioate yielded new conjugates 6a and 6b respectively. In addition, the compounds 5c and 6a,b have been evaluated for their in vitro cytotoxicity on some of the selected human cancer cell lines and these conjugates have exhibited significant cytotoxic activity. Further, the DNA-binding ability of these new conjugates has been evaluated by using thermal denaturation (ΔTm) studies. The correlation between structure and DNA-binding ability has been investigated by molecular modeling studies which predicted that 6b exhibits superior DNA-binding ability and these are in agreement with the experimental DNA-binding studies. PMID:25665519

  7. HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND THE ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS: BINDING OF GLICLAZIDE WITH GLYCATED HUMAN SERUM ALBUMIN

    OpenAIRE

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K S; Hage, David S.

    2011-01-01

    This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclaz...

  8. X-ray Crystal Structure of the Novel Enhanced-Affinity Glucocorticoid Agonist Fluticasone Furoate in the Glucocorticoid Receptor−Ligand Binding Domain

    Energy Technology Data Exchange (ETDEWEB)

    Biggadike, Keith; Bledsoe, Randy K.; Hassell, Anne M.; Kirk, Barrie E.; McLay, Iain M.; Shewchuk, Lisa M.; Stewart, Eugene L. (GSKNC); (GSK)

    2008-07-08

    An X-ray crystal structure is reported for the novel enhanced-affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand binding domain of the glucocorticoid receptor. Comparison of this structure with those of dexamethasone and fluticasone propionate shows the 17{alpha} furoate ester to occupy more fully the lipophilic 17{alpha} pocket on the receptor, which may account for the enhanced glucocorticoid receptor binding of FF.

  9. Predicting protein ligand binding motions with the conformation explorer

    Directory of Open Access Journals (Sweden)

    Flores Samuel C

    2011-10-01

    Full Text Available Abstract Background Knowledge of the structure of proteins bound to known or potential ligands is crucial for biological understanding and drug design. Often the 3D structure of the protein is available in some conformation, but binding the ligand of interest may involve a large scale conformational change which is difficult to predict with existing methods. Results We describe how to generate ligand binding conformations of proteins that move by hinge bending, the largest class of motions. First, we predict the location of the hinge between domains. Second, we apply an Euler rotation to one of the domains about the hinge point. Third, we compute a short-time dynamical trajectory using Molecular Dynamics to equilibrate the protein and ligand and correct unnatural atomic positions. Fourth, we score the generated structures using a novel fitness function which favors closed or holo structures. By iterating the second through fourth steps we systematically minimize the fitness function, thus predicting the conformational change required for small ligand binding for five well studied proteins. Conclusions We demonstrate that the method in most cases successfully predicts the holo conformation given only an apo structure.

  10. Insights into affinity and specificity in the complexes of α-lytic protease and its inhibitor proteins: binding free energy from molecular dynamics simulation†

    Science.gov (United States)

    Cieplak, Piotr

    2014-01-01

    We report the binding free energy calculation and its decomposition for the complexes of α-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have vastly different affinity towards α-lytic protease (ALP), a bacterial serine protease. OMTKY3 inhibits the enzyme much more weakly (by ~106 times) than eglin C. Moreover, a variant of OMTKY3 with five mutations, OMTKY3M, has been shown to inhibit 104 times more strongly than the wild-type inhibitor. The underlying mechanisms for the unusually large difference in binding affinities and the effect of mutation are not well understood. Here we use molecular dynamics simulation with molecular mechanics–Poisson Boltzmann/surface area method (MM-PB/SA) to investigate quantitatively the binding specificity. The calculated absolute binding free energies correctly differentiate the thermodynamic stabilities of these protein complexes, but the magnitudes of the binding affinities are systematically overestimated. Analysis of the binding free energy components provides insights into the molecular mechanism of binding specificity. The large ΔΔGbind between eglin C and wild type OMTKY3 towards ALP is mainly attributable to the stronger nonpolar interactions in the ALP-eglin C complex, arising from a higher degree of structural complementarity. Here the electrostatic interaction contributes to a lesser extent. The enhanced inhibition in the penta-mutant OMTKY3M over its wild type is entirely due to an overall improvement in the solvent-mediated electrostatic interactions in the ALP-OMTKY3M complex. The results suggest that for these protein-complexes and similar enzyme-inhibitor systems

  11. CYCLIC CHOLECYSTOKININ ANALOGUES EXHIBIT HIGH BLOOD STABILITY AND BINDING AFFINITY WITH CHOLECYSTOKININ RECEPTOR

    Directory of Open Access Journals (Sweden)

    Eun-Ha Joh

    2014-01-01

    Full Text Available Recently, incidence of Cholecystokinin (CCK receptor is recognized as a factor that determines the aggressive phenotype of pancreatic cancer. In this study, a novel Cholecystokinin (CCK analogues; DOTA-Nle-cyclo (Glu-Trp-Met-Asp-Phe-Lys-NH2 (DOTA-cCCK and DOTA-Nle-cyclo (Glu-Trp-Nle-Asp-Phe-Lys-NH2 (DOTA-[Nle]-cCCK were synthesized and radiolabeled and the targeting abilities on the CCK receptor were evaluated for new CCK receptor targeting agents searching. Peptides were prepared through a solid phase synthesis method and their purity was over 98%. DOTA is the chelating agent for 68Ga-labelling, which the peptides were radiolabeled with 68Ga by a high radiolabeling yield (>98%. Peptides were stable over 98% by incubation in mouse blood at 37°C for 2 h. A competitive displacement of 125I-CCK8 on the AR42J human pancreatic carcinoma cells revealed that 50% inhibitory concentration value (IC50 were 12.31 nM of DOTA-cCCK and 1.69 nM of DOTA-[Nle]-cCCK. Stable in the blood of both DOTA-cCCK and DOTA-[Nle]-cCCK, but the binding rate with the CCK receptor on AR42J cells, DOTA-[Nle]-cCCK confirmed better than DOTA-cCCK. Therefore, it is concluded that 68Ga-DOTA-[Nle]-cCCK can be potential candidate as a targeting modality for the CCK receptor over-expressing tumors and further studies to evaluate their biological characteristics are needed.

  12. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    DEFF Research Database (Denmark)

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    . This result shows that in addition to D1, which has an established function in ligand binding (Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266, 7842-7847), D3 has an important role in governing a high affinity in the intact receptor. Real-time biomolecular...

  13. Automated benchmarking of peptide-MHC class I binding predictions

    DEFF Research Database (Denmark)

    Trolle, Thomas; Metushi, Imir G.; Greenbaum, Jason;

    2015-01-01

    Motivation: Numerous in silico methods predicting peptide binding to major histocompatibility complex (MHC) class I molecules have been developed over the last decades. However, the multitude of available prediction tools makes it non-trivial for the end-user to select which tool to use for a giv......-user to make educated selections between participating tools. Of the four participating servers, NetMHCpan performed the best, followed by ANN, SMM and finally ARB. Availability and implementation: Up-to-date performance evaluations of each server can be found online at http...... the public access to frequent, up-to-date performance evaluations of all participating tools. To overcome potential selection bias in the data included in the IEDB, a strategy was implemented that suggests a set of peptides for which different prediction methods give divergent predictions as to their...

  14. Multispectroscopic and docking studies on the binding of chlorogenic acid isomers to human serum albumin: Effects of esteryl position on affinity.

    Science.gov (United States)

    Tang, Bin; Huang, Yanmei; Ma, Xiangling; Liao, Xiaoxiang; Wang, Qing; Xiong, Xinnuo; Li, Hui

    2016-12-01

    Structural differences among various dietary polyphenols affect their absorption, metabolism, and bioactivities. In this work, chlorogenic acid (CA) and its two positional isomers, neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA), were investigated for their binding reactions with human serum albumin (HSA) using fluorescence, ultraviolet-visible, Fourier transform infrared and circular dichroism spectroscopies, as well as molecular docking. All three isomers were bound to HSA at Sudlow's site I and affected the protein secondary structure. CCA presented the strongest ability of hydrogen-bond formation, and both CA and NCA generated more electrostatic interactions with HSA. The albumin-binding capacity of these compounds decreased in the order CCA>NCA>CA. The compound with 4-esteryl structure showed higher binding affinity and larger conformational changes to HSA than that with 3- or 5-esteryl structures. These comparative studies on structure-affinity relationship contributed to the structural modification and design of phenolic food additives or new polyphenol-like drugs. PMID:27374553

  15. Novel Radioiodinated γ-Hydroxybutyric Acid Analogues for Radiolabeling and Photolinking of High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    homogenate and slices. Our data show that [125I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([125I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (Kd, 7 nM; Bmax, 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ...... cortical and hippocampal membranes. [125I]BnOPh-GHB is the first reported 125I-labeled GHB radioligand and is a useful tool for in vitro studies of the specific high-affinity GHB binding sites. The related photoaffinity linker [125I]4-hydroxy-4-[4-(2-azido-5-iodobenzyloxy)phenyl]butanoate can be used as a...

  16. Development and optimization of a competitive binding assay for the galactophilic low affinity lectin LecA from Pseudomonas aeruginosa.

    Science.gov (United States)

    Joachim, Ines; Rikker, Sebastian; Hauck, Dirk; Ponader, Daniela; Boden, Sophia; Sommer, Roman; Hartmann, Laura; Titz, Alexander

    2016-08-16

    Infections with the Gram-negative bacterium Pseudomonas aeruginosa result in a high mortality among immunocompromised patients and those with cystic fibrosis. The pathogen can switch from planktonic life to biofilms, and thereby shields itself against antibiotic treatment and host immune defense to establish chronic infections. The bacterial protein LecA, a C-type lectin, is a virulence factor and an integral component for biofilm formation. Inhibition of LecA with its carbohydrate ligands results in reduced biofilm mass, a potential Achilles heel for treatment. Here, we report the development and optimization of a fluorescence polarization-based competitive binding assay with LecA for application in screening of potential inhibitors. As a consequence of the low affinity of d-galactose for LecA, the fluorescent ligand was optimized to reduce protein consumption in the assay. The assay was validated using a set of known inhibitors of LecA and IC50 values in good agreement with the known Kd values were obtained. Finally, we employed the optimized assay to screen sets of synthetic thio-galactosides and natural blood group antigens and report their structure-activity relationship. In addition, we evaluated a multivalent fluorescent assay probe for LecA and report its applicability in an inhibition assay. PMID:27488655

  17. ‘maskBAD’ – a package to detect and remove Affymetrix probes with binding affinity differences

    Directory of Open Access Journals (Sweden)

    Dannemann Michael

    2012-04-01

    Full Text Available Abstract Background Hybridization differences caused by target sequence differences can be a confounding factor in analyzing gene expression on microarrays, lead to false positives and reduce power to detect real expression differences. We prepared an R Bioconductor compatible package to detect, characterize and remove such probes in Affymetrix 3’IVT and exon-based arrays on the basis of correlation of signal intensities from probes within probe sets. Results Using completely mouse genomes we determined type 1 (false negatives and type 2 (false positives errors with high accuracy and we show that our method routinely outperforms previous methods. When detecting 76.2% of known SNP/indels in mouse expression data, we obtain at most 5.5% false positives. At the same level of false positives, best previous method detected 72.6%. We also show that probes with differing binding affinity both hinder differential expression detection and introduce artifacts in cancer-healthy tissue comparison. Conclusions Detection and removal of such probes should be a routine step in Affymetrix data preprocessing. We prepared a user friendly R package, compatible with Bioconductor, that allows the filtering and improving of data from Affymetrix microarrays experiments.

  18. Prediction of solid–binder affinity in dry and aqueous systems: Work of adhesion approach vs. ideal tensile strength approach

    OpenAIRE

    Jarray, Ahmed; Gerbaud, Vincent; Hemati, Mehrdji

    2015-01-01

    Wet granulation process requiresthe addition of a coating agentor binder, typicallycomposed of surfactants such as hydroxypropyl-methylcellulose (HPMC), water and a small amount offiller such as stearic acid (SA). In dry granulation however, the coating agent is added to the system in the form offine solid particles. In both cases, a successful granulation requires good affinity between host and guest particles. In this study, we compare two approaches to predict the binder–substr...

  19. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-Cβ antibody

    International Nuclear Information System (INIS)

    Highlights: ► A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. ► TCR-Fc protein immobilized by an anti-Cβ antibody bound to a p/MHC tetramer. ► Binding affinity of TCR-Fc was markedly increased by binding with anti-Cβ antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100–200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-Cβ antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 × 10−5 M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-Cβ antibody, its binding affinity for p/MHC increased by 5-fold (2.2 × 10−6 M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-Cβ antibody, which is probably due to the stabilization of the Vα/Vβ region of the TCR. These findings provide new insights into the binding of sTCRs to p/MHCs and will hopefully be instrumental in establishing

  20. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jin, Aishun [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Immunology, College of Basic Medical Sciences, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081 (China); Kishi, Hiroyuki, E-mail: immkishi@med.u-toyama.ac.jp [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Muraguchi, Atsushi [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

  1. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

    1988-05-05

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. (Phe/sup -1/, Val/sup 1/, Asn/sup 2/, Gln/sup 3/, His/sup 4/, Ser/sup 8/, His/sup 9/, Glu/sup 12/, Tyr/sup 15/, Leu/sup 16/)IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. (Gln/sup 3/, Ala/sup 4/) IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. (Tyr/sup 15/, Leu/sup 16/) IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, (Gln/sup 3/, Ala/sup 4/, Tyr/sup 15/,Leu/sup 16/)IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.

  2. Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins.

    Science.gov (United States)

    Jiao, Chensong; Summerlin, Matthew; Bruzik, Karol S; Hanakahi, Leslyn

    2015-10-20

    Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) in vitro. The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin. IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2, which is known to bind IP6, and nucleolin. Protein binding to IP6-biotin is selective, as IP3, IP4, and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool for investigating the role of IP6 in biological systems. PMID:26397942

  3. The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

    Directory of Open Access Journals (Sweden)

    van Nuland Nico AJ

    2007-04-01

    Full Text Available Abstract Background SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41 binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3, while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3. Results Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several

  4. High affinity binding of the heat-stable protein kinase inhibitor to the catalytic subunit of cAMP-dependent protein kinase is selectively abolished by mutation of Arg133.

    Science.gov (United States)

    Wen, W; Taylor, S S

    1994-03-18

    The two classes of physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase are the regulatory subunits and the heat-stable protein kinase inhibitors (PKIs), and both share a common mechanism of inhibition. Each has a similar inhibitor site that resembles a peptide substrate, and this occupies the P-3 to P+1 portion of the peptide recognition site. However, in addition to this consensus site, each inhibitor requires a peripheral binding site to achieve high affinity binding. Arg134 and Arg133 lie on the surface of the catalytic subunit with Arg133 coming close to the amphipathic helix of PKI(5-24) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N.-h., Taylor, S. S., and Sowadski, J. M. (1991) Science 253, 414-420). Replacement of Arg134 and Arg133 with Ala selectively abolishes the high affinity binding of PKI. Replacement of Arg133 alone is sufficient to give the same phenotype. In the presence of MgATP, the Kd,app, is increased from < 0.2 to 105 nM and, in the absence of ATP, the Kd is too large to be reliably measured. Based on the crystal structure, Arg133 hydrogen bonds to the P-7 backbone carbonyl of PKI(5-24). However, more importantly, it also contributes to the hydrophobicity of the P-11 binding site in the C.PKI(5-24) complex. We predict that it is the perturbation of this hydrophobic pocket that accounts for the effects of this mutation. In the absence of peptide, Arg133 may help to stabilize Glu230, a buried carboxylate that binds to the P-2 Arg in the crystal structure of C.PKI(5-24). Replacement of Arg133 and Arg134 with Ala has little effect on catalysis using a heptapeptide substrate and has no effect on the inhibition of the catalytic subunit by the regulatory subunit. The results thus demonstrate that these two inhibitor proteins that both bind to the catalytic subunit with a high affinity utilize different sites on the enzyme to achieve tight binding. The gamma isoform of the catalytic subunit is insensitive to

  5. The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity

    DEFF Research Database (Denmark)

    Hansen, B E; Nielsen, Claus Henrik; Madsen, H O;

    2011-01-01

    carrying the strongest genetic association with MS. In contrast with HLA-DR and -DQ molecules, HLA-DP molecules are poorly characterized with respect to the binding of self-peptides. We show here that HLA-DP2 binds MBP85-99 with high affinity, and that the amino acid residues in position MBP91, MBP92 and...... MBP93 are influencing the binding, as shown by alanine scans. We further used a series of truncated peptides to identify the core of the binding. Moving the frame along the peptide from residues 87-97 to 89-99 progressively decreased the binding affinity for HLA-DP2, while moving further towards the C......-terminal completely abrogated the binding of peptides to HLA-DP2. The data suggest that the docking of the MBP85-99 peptide into the HLA-DP2 groove is dependent on MBP88V and MBP89V and may use either of them as primary anchor for the p1 position. HLA-DP2 might thus present the MBP85-99 peptide in the same register...

  6. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    International Nuclear Information System (INIS)

    Binding studies were performed with two 125I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other δ-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis δ-endotoxins active against M. sexta compete for binding of 125I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles

  7. Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors

    Institute of Scientific and Technical Information of China (English)

    Paul C. SEHNKE; Beth J. LAUGHNER; Carla R. LYERLY LINEBARGER; William B. GURLEY; Robert J.FERL

    2005-01-01

    Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs).The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood.In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A.thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

  8. Allosteric effects of R- and S-citalopram on the human 5-HT transporter: evidence for distinct high- and low-affinity binding sites

    DEFF Research Database (Denmark)

    Plenge, Per; Gether, Ulrik; Rasmussen, Søren G

    2007-01-01

    cells, and their 5-HT uptake and uptake inhibitor-binding abilities were studied. The hSERT mutations did not alter affinities for 5-HT or paroxetine, but high-affinity binding of S-citalopram was severely affected, particularly by the I172M, and Y95F/I172M mutations - K(i) respectively 4 nM (wild......-type), 35 nM, 1000 nM, and 17.100 nM (mutants). The allosteric site however, in wild-type hSERT and the three mutants was unaffected by the mutations as attenuation of the dissociation rate of the [(3)H]-paroxetine:hSERT complex in the presence of S-citalopram or paroxetine was the same for wild-type h...

  9. The development of an affinity evaluation and prediction system by using protein–protein docking simulations and parameter tuning

    Directory of Open Access Journals (Sweden)

    Koki Tsukamoto

    2009-01-01

    Full Text Available Koki Tsukamoto1, Tatsuya Yoshikawa1,2, Kiyonobu Yokota1, Yuichiro Hourai1, Kazuhiko Fukui11Computational Biology Research Center (CBRC, National Institute of Advanced Industrial Science and Technology (AIST, Koto-ku, Tokyo, Japan; 2Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Toyonaka, Osaka, JapanAbstract: A system was developed to evaluate and predict the interaction between protein pairs by using the widely used shape complementarity search method as the algorithm for docking simulations between the proteins. We used this system, which we call the affinity evaluation and prediction (AEP system, to evaluate the interaction between 20 protein pairs. The system first executes a “round robin” shape complementarity search of the target protein group, and evaluates the interaction between the complex structures obtained by the search. These complex structures are selected by using a statistical procedure that we developed called ‘grouping’. At a prevalence of 5.0%, our AEP system predicted protein–protein interactions with a 50.0% recall, 55.6% precision, 95.5% accuracy, and an F-measure of 0.526. By optimizing the grouping process, our AEP system successfully predicted 10 protein pairs (among 20 pairs that were biologically relevant combinations. Our ultimate goal is to construct an affinity database that will provide cell biologists and drug designers with crucial information obtained using our AEP system.Keywords: protein–protein interaction, affinity analysis, protein–protein docking, FFT, massive parallel computing

  10. Cyclic Lactam Hybrid α-MSH/Agouti-Related Protein (AGRP) Analogues with Nanomolar Range Binding Affinities at the Human Melanocortin Receptors

    OpenAIRE

    Mayorov, Alexander V.; Cai, Minying; Palmer, Erin S.; Tanaka, Dustin K.; Cain, James P.; Dedek, Matthew M.; Tan, Bahar; Trivedi, Dev; Victor J. Hruby

    2011-01-01

    A novel hybrid melanocortin pharmacophore was designed based on the topographical similarities between the pharmacophores of Agouti related protein (AGRP) an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. When employed in two different 23-membered macrocyclic lactam peptide templates, the designed hybrid AGRP/MSH pharmacophore yielded non-competitive ligands with nanomolar range binding affinities. The topography-based pha...

  11. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse

    OpenAIRE

    Torres, Oscar B.; Antoline, Joshua F. G.; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C.; Alving, Carl R.; Matyas, Gary R.

    2015-01-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antib...

  12. Encapsulation of Vitamin B(1) and Its Phosphate Derivatives by Cucurbit[7]uril: Tunability of the Binding Site and Affinity by the Presence of Phosphate Groups.

    Science.gov (United States)

    Li, Shengke; Yin, Hang; Wyman, Ian W; Zhang, Qingwen; Macartney, Donal H; Wang, Ruibing

    2016-02-01

    Vitamin B1 (1) and its phosphate derivatives, thiamine monophosphate (2) and thiamine pyrophosphate (3), are shown to form stable 1:1 host-guest complexes with cucurbit[7]uril (CB[7]) in aqueous solution. The binding sites of CB[7] on these guests shift from the ethylthiazolium region of 1 to the pyrimidine moiety of 2 and 3 due to the presence of phosphate groups, leading to variations of binding affinities as well as C(2)-H/D exchange rate constants and C(2)-H pKa values with these guest molecules. PMID:26745735

  13. High affinity and temperature sensitivity of blood oxygen binding in Pangasianodon hypophthalmus due to lack of chloride-hemoglobin allosteric interaction

    DEFF Research Database (Denmark)

    Damsgaard, Christian; Phuong, Le My; Huong, Do Thi Thanh; Jensen, Frank Bo; Wang, Tobias; Bayley, Mark

    2015-01-01

    Air-breathing fishes represent interesting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and, further, are of great socio-economic importance for aquaculture in Southeast Asia. To understand how environmental factors, such as...... high temperature, affect O2 transport in air-breathing fishes, this study assessed the effects of temperature on O2 binding of blood and Hb in the economically important air-breathing fish Pangasianodon hypophthalmus. To determine blood O2 binding properties, blood was drawn from resting cannulated...... fishes and O2 binding curves made at 25°C and 35°C. To determine the allosteric regulation and thermodynamics of Hb O2 binding, Hb was purified, and O2 equilibria were recorded at five temperatures in the absence and presence of ATP and Cl-. Whole blood had a high O2 affinity (O2 tension at half...

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

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

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

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

    International Nuclear Information System (INIS)

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB1Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB2Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB2Rs (hCB2Rs). The affinity of cannabinoids for hCB2Rs was determined by competition binding studies employing CHO-hCB2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB2Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB2Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ9-tetrahydrocannabinol (Δ9-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB2R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB2Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB2Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB1 and CB2Rs. - Highlights: • JWH-018 and JWH-073 are synthetic cannabinoids present in abused K2 products. • JWH-018, JWH-073 and

  16. Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.

    Science.gov (United States)

    Li, Qian; Zhang, Tianlong; Bian, Liujiao

    2016-03-01

    Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties. PMID:26882128

  17. Modulation of DNA-polyamide interaction by β-alanine substitutions: a study of positional effects on binding affinity, kinetics and thermodynamics.

    Science.gov (United States)

    Wang, Shuo; Aston, Karl; Koeller, Kevin J; Harris, G Davis; Rath, Nigam P; Bashkin, James K; Wilson, W David

    2014-10-14

    Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and frequently employ a flexible motif, β-alanine (β), to reduce the molecular rigidity to maintain the DNA recognition register. To better understand the diverse effects that β can have on DNA-PA binding affinity, selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for an eight-heterocyclic hairpin PA and its β derivatives with their cognate and mutant sequences. With these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double βs. A set of complementary experiments have been conducted to evaluate the molecular interactions in detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration calorimetry. The β substitutions generally weaken the binding affinities of these PAs with cognate DNA, and have large and diverse influences on PA binding kinetics in a position- and number-dependent manner. The DNA base mutations have also shown positional effects on the binding of a single PA. Besides the β substitutions, the monocationic Dp group [3-(dimethylamino)propylamine] in parent PA has been modified into a dicationic Ta group (3,3'-diamino-N-methyldipropylamine) to minimize the frequently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first time. This combined set of results significantly extends our understanding of the energetic basis of specific DNA recognition by PAs. PMID:25141096

  18. Conformational destabilization of Immunoglobulin G increases the low pH-binding affinity with the Neonatal Fc Receptor

    DEFF Research Database (Denmark)

    Walters, Benjamin T; Jensen, Pernille Foged; Larraillet, Vincent;

    2016-01-01

    Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the...... diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and Fc...

  19. Inactivation of Na,K-ATPase following Co(NH3)4ATP binding at a low affinity site in the protomeric enzyme unit.

    Science.gov (United States)

    Ward, Douglas G; Cavieres, José D

    2003-04-25

    The Na(+)-dependent or E1 stages of the Na,K-ATPase reaction require a few micromolar ATP, but submillimolar concentrations are needed to accelerate the K(+)-dependent or E2 half of the cycle. Here we use Co(NH(3))(4)ATP as a tool to study ATP sites in Na,K-ATPase. The analogue inactivates the K(+) phosphatase activity (an E2 partial reaction) and the Na,K-ATPase activity in parallel, whereas ATP-[(3)H]ADP exchange (an E1 reaction) is affected less or not at all. Although the inactivation occurs as a consequence of low affinity Co(NH(3))(4)ATP binding (K(D) approximately 0.4-0.6 mm), we can also measure high affinity equilibrium binding of Co(NH(3))(4)[(3)H]ATP (K(D) = 0.1 micro m) to the native enzyme. Crucially, we find that covalent enzyme modification with fluorescein isothiocyanate (which blocks E1 reactions) causes little or no effect on the affinity of the binding step preceding Co(NH(3))(4)ATP inactivation and only a 20% decrease in maximal inactivation rate. This suggests that fluorescein isothiocyanate and Co(NH(3))(4)ATP bind within different enzyme pockets. The Co(NH(3))(4)ATP enzyme was solubilized with C(12)E(8) to a homogeneous population of alphabeta protomers, as verified by analytical ultracentrifugation; the solubilization did not increase the Na,K-ATPase activity of the Co(NH(3))(4)ATP enzyme with respect to parallel controls. This was contrary to the expectation for a hypothetical (alphabeta)(2) membrane dimer with a single ATP site per protomer, with or without fast dimer/protomer equilibrium in detergent solution. Besides, the solubilized alphabeta protomer could be directly inactivated by Co(NH(3))(4)ATP, to less than 10% of the control Na,K-ATPase activity. This suggests that the inactivation must follow Co(NH(3))(4)ATP binding at a low affinity site in every protomeric unit, thus still allowing ATP and ADP access to phosphorylation and high affinity ATP sites. PMID:12591931

  20. Affinity Chromatography Method for Determination of Binding of Drugs to Melanin and Evaluation of Side Effect Potential of Antipsychotic Agents

    OpenAIRE

    Marszałł, Michał Piotr; Proszowska, Anna; Buciński, Adam; Kaliszan, Roman

    2014-01-01

    The extrapyramidal side effect parameters of typical and atypical antypsychotics were correlated with affinity chromatographic data determined on the melanin-based column. The chromatographic study was performed according to the hypothesis that extrapyramidal symptoms (EPS) as side effects of the use of antipsychotic drugs at clinically effective doses are correlated to the affinity of these drugs to neuromelanin. For that aim the polymerization product of L-DOPA (melanin) was immobilized ont...

  1. Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis.

    Science.gov (United States)

    Rudra, Suparna; Dasmandal, Somnath; Patra, Chiranjit; Kundu, Arjama; Mahapatra, Ambikesh

    2016-09-01

    The binding interaction of a synthesized Schiff base Fe(II) complex with biological macromolecules viz., bovine serum albumin (BSA) and calf thymus(ct)-DNA have been investigated using different spectroscopic techniques coupled with viscosity measurements at physiological pH and 298K. Regular amendments in emission intensities of BSA upon the action of the complex indicate significant interaction between them, and the binding interaction have been characterized by Stern Volmer plots and thermodynamic binding parameters. On the basis of this quenching technique one binding site with binding constant (Kb=(7.6±0.21)×10(5)) between complex and protein have been obtained at 298K. Time-resolved fluorescence studies have also been encountered to understand the mechanism of quenching induced by the complex. Binding affinities of the complex to the fluorophores of BSA namely tryptophan (Trp) and tyrosine (Tyr) have been judged by synchronous fluorescence studies. Secondary structural changes of BSA rooted by the complex has been revealed by CD spectra. On the other hand, hypochromicity of absorption spectra of the complex with the addition of ct-DNA and the gradual reduction in emission intensities of ethidium bromide bound ct-DNA in presence of the complex indicate noticeable interaction between ct-DNA and the complex with the binding constant (4.2±0.11)×10(6)M(-1). Life-time measurements have been studied to determine the relative amplitude of binding of the complex to ct-DNA base pairs. Mode of binding interaction of the complex with ct-DNA has been deciphered by viscosity measurements. CD spectra have also been used to understand the changes in ct-DNA structure upon binding with the metal complex. Density functional theory (DFT) and molecular docking analysis have been employed in highlighting the interactive phenomenon and binding location of the complex with the macromolecules. PMID:27214273

  2. Combined molecular dynamics and continuum solvent approaches (MM-PBSA/GBSA) to predict noscapinoid binding to γ-tubulin dimer.

    Science.gov (United States)

    Suri, C; Naik, P K

    2015-06-01

    γ-tubulin plays crucial role in the nucleation and organization of microtubules during cell division. Recent studies have also indicated its role in the regulation of microtubule dynamics at the plus end of the microtubules. Moreover, γ-tubulin has been found to be over-expressed in many cancer types, such as carcinomas of the breast and glioblastoma multiforme. These studies have led to immense interest in the identification of chemical leads that might interact with γ-tubulin and disrupt its function in order to explore γ-tubulin as potential chemotherapeutic target. Recently a colchicine-interacting cavity was identified at the interface of γ-tubulin dimer that might also interact with other similar compounds. In the same direction we theoretically investigated binding of a class of compounds, noscapinoids (noscapine and its derivatives) at the interface of the γ-tubulin dimer. Molecular interaction of noscapine and two of its derivatives, amino-noscapine and bromo-noscapine, was investigated by molecular docking, molecular dynamics simulation and binding free energy calculation. All noscapinoids displayed stable interaction throughout simulation of 25 ns. The predictive binding free energy (ΔGbind) indicates that noscapinoids bind strongly with the γ-tubulin dimer. However, bromo-noscapine showed the best binding affinity (ΔGbind = -37.6 kcal/mol) followed by noscapine (ΔGbind = -29.85 kcal/mol) and amino-noscapine (ΔGbind = -23.99 kcal/mol) using the MM-PBSA method. Similarly using the MM-GBSA method, bromo-noscapine showed highest binding affinity (ΔGbind = -43.64 kcal/mol) followed by amino-noscapine (ΔGbind = -37.56 kcal/mol) and noscapine (ΔGbind = -34.57 kcal/mol). The results thus generate compelling evidence that these noscapinoids may hold great potential for preclinical and clinical evaluation. PMID:26274780

  3. Convolutional neural network architectures for predicting DNA–protein binding

    Science.gov (United States)

    Zeng, Haoyang; Edwards, Matthew D.; Liu, Ge; Gifford, David K.

    2016-01-01

    Motivation: Convolutional neural networks (CNN) have outperformed conventional methods in modeling the sequence specificity of DNA–protein binding. Yet inappropriate CNN architectures can yield poorer performance than simpler models. Thus an in-depth understanding of how to match CNN architecture to a given task is needed to fully harness the power of CNNs for computational biology applications. Results: We present a systematic exploration of CNN architectures for predicting DNA sequence binding using a large compendium of transcription factor datasets. We identify the best-performing architectures by varying CNN width, depth and pooling designs. We find that adding convolutional kernels to a network is important for motif-based tasks. We show the benefits of CNNs in learning rich higher-order sequence features, such as secondary motifs and local sequence context, by comparing network performance on multiple modeling tasks ranging in difficulty. We also demonstrate how careful construction of sequence benchmark datasets, using approaches that control potentially confounding effects like positional or motif strength bias, is critical in making fair comparisons between competing methods. We explore how to establish the sufficiency of training data for these learning tasks, and we have created a flexible cloud-based framework that permits the rapid exploration of alternative neural network architectures for problems in computational biology. Availability and Implementation: All the models analyzed are available at http://cnn.csail.mit.edu. Contact: gifford@mit.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307608

  4. Characterization of a high affinity interleukin-1 (IL-1) specific binding site in a human synovial sarcoma (Hs431) cell line

    International Nuclear Information System (INIS)

    A subpopulation of cells was derived from the Hs431 connective tissue sarcoma cell line which possessed high affinity (estimated Kd = 0.38-0.55 nM) binding sites for human recombinant [125I]-IL-1 alpha. Binding at 4 degrees C was slow approaching equilibrium by 4 hrs. Dissociation of [125I]-IL-1 alpha was also slow and unaffected by high concentrations of cold ligand. The binding site also underwent ligand-induced internalization at 37 degrees C. An Mr = 83,000 protein was identified in affinity crosslinking studies. Despite these similarities to previously reported IL-1 receptors, Hs431 cells did not exhibit biological responses to IL-1 which have been observed in other cell lines. IL-1 did not induce PGE2 or collagenase synthesis. IL-1 also failed to induce ornithine decarboxylase activity (ODC) or stimulate [3H]-thymidine incorporation. In contrast, the Hs431 cells did contain a functional epidermal growth factor (EGF) receptor as determined from binding studies, protein kinase activity, induction of ODC, and stimulation of [3H]-thymidine incorporation. Thus, the refractoriness of Hs431 cells to IL-1 was fairly specific and did not result from a generalized defect associated with cell transformation

  5. A high-affinity soluble folate receptor in fluids of non-neoplastic ovarian cysts. Radioligand binding, molecular size, hydrophobic residue, and immunological properties

    International Nuclear Information System (INIS)

    The presence of a soluble folate receptor in fluids of non-neoplastic ovarian cysts was demonstrated. Radioligand binding exhibited characteristics typical of high-affinity folate-binding proteins. These included positive cooperativity, a tendency to increased binding affinity with decreasing receptor concentration, a slow ligand dissociation at pH 7.4 and inhibition by folate analogues. The folate receptor was probably synthesized in the lining epithelial cells of the cysts which showed positive immunostaining with antibodies against human milk folate-binding protein. The gel filtration profile of cystic fluid contained two radioligand-bound peaks, 25 and 100 kDa, whereas a single band of 70 kDa was seen on SDS-PAGE immunoblotting. Treatment with the enzyme phosphatidyl-inositol-specific phospholipase C resulted in a partial conversion of the 100 kDa peak to the 25 kDa peak. This suggests that insertion of a hydrophobic glycosyl-phosphatidyl-inositol tail into Triton X-100 micelles could give rise to large molecular size forms of the receptor on gel filtration. (au) 30 refs

  6. The characterization of high-affinity binding sites in rat brain for the mast cell-degranulating peptide from bee venom using the purified monoiodinated peptide.

    Science.gov (United States)

    Taylor, J W; Bidard, J N; Lazdunski, M

    1984-11-25

    The preparation of a pure, monoiodinated derivative of mast cell-degranulating peptide (MCD peptide), the mast cell-degranulating peptide from bee venom, has enabled us to identify binding sites in rat brain membranes that have a high affinity and specificity for this peptide. These binding sites are evenly distributed throughout the brain and copurify with synaptic membranes. Saturation-binding curves, determined by rapid centrifugation or filtration assays, indicate a single population of sites with a concentration of 200 fmol/mg membrane protein in partially fractionated, lysed brain membranes. Dissociation constants of 150 and 140 pM were calculated for the iodinated and native peptides, respectively. These binding sites are probably associated with the neurotoxic action of MCD peptide in the central nervous system. No similar binding sites have been identified in peripheral tissue preparations, and other polycationic mast cell-degranulating agents including compound 48/80 show no such specificity. Specific modification of the primary amines, arginine residues, or disulfide bridges of MCD peptide results in a complete loss of binding activity. Other components of bee venom show specificity for the MCD peptide-binding site, suggesting that a class of neurotoxins in bee venom (possibly including secapin and tertiapin, but not apamin) share the specific action of MCD peptide on the central nervous system. PMID:6501283

  7. Hot spots and transient pockets: predicting the determinants of small-molecule binding to a protein-protein interface.

    Science.gov (United States)

    Metz, Alexander; Pfleger, Christopher; Kopitz, Hannes; Pfeiffer-Marek, Stefania; Baringhaus, Karl-Heinz; Gohlke, Holger

    2012-01-23

    Protein-protein interfaces are considered difficult targets for small-molecule protein-protein interaction modulators (PPIMs ). Here, we present for the first time a computational strategy that simultaneously considers aspects of energetics and plasticity in the context of PPIM binding to a protein interface. The strategy aims at identifying the determinants of small-molecule binding, hot spots, and transient pockets, in a protein-protein interface in order to make use of this knowledge for predicting binding modes of and ranking PPIMs with respect to their affinity. When applied to interleukin-2 (IL-2), the computationally inexpensive constrained geometric simulation method FRODA outperforms molecular dynamics simulations in sampling hydrophobic transient pockets. We introduce the PPIAnalyzer approach for identifying transient pockets on the basis of geometrical criteria only. A sequence of docking to identified transient pockets, starting structure selection based on hot spot information, RMSD clustering and intermolecular docking energies, and MM-PBSA calculations allows one to enrich IL-2 PPIMs from a set of decoys and to discriminate between subgroups of IL-2 PPIMs with low and high affinity. Our strategy will be applicable in a prospective manner where nothing else than a protein-protein complex structure is known; hence, it can well be the first step in a structure-based endeavor to identify PPIMs. PMID:22087639

  8. Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors

    Directory of Open Access Journals (Sweden)

    Sathyanarayana Bangalore K

    2006-10-01

    Full Text Available Abstract Background The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment. Results In this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent Kd of 5–10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is observed even after 10 sec. A five-fold molar excess of soluble MUC16 was unable to completely inhibit the binding of mesothelin to the OVCAR-3 cells. Oxidation of the MUC16 glycans, removal of its N-linked oligosaccharides, and treatment of the mucin with wheat germ agglutinin and erythroagglutinating phytohemagglutinin abrogates its binding to mesothelin. These observations suggest that at least a subset of the MUC16-asscociated N-glycans is required for binding to mesothelin. We also demonstrate that MUC16 positive ovarian tumor cells exhibit increased adherence to A431 cells transfected with mesothelin (A431-Meso+. Only minimal adhesion is observed between MUC16 knockdown cells and A431-Meso+ cells. The binding between the MUC16 expressing ovarian tumor cells and the A431-Meso+ cells occurs even in the presence of ascites from patients with ovarian cancer. Conclusion The strong binding kinetics of the mesothelin-MUC16

  9. Glial high-affinity binding site with specificity for angiotensin II not angiotensin III: a possible N-terminal-specific converting enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Printz, M.P.; Jennings, C.; Healy, D.P.; Kalter, V.

    1986-01-01

    Anomalous binding properties of angiotensin II to fetal rat brain primary cultures suggested a possible contribution from contaminating glia. To investigate this possibility, cultures of C6 glioma, a clonal rat cell line, were examined for the presence of angiotensin II receptors. A specific high-affinity site for (/sup 125/I)angiotensin II was measured both by traditional methodology using whole cells and by autoradiography. This site shared properties similar to that found with the brain cells, namely low ligand internalization and markedly decreased affinity for N-terminal sarcosine or arginine-angiotensin analogs. The competition rank order was angiotensin II much greater than (Sar1,Ile8)angiotensin II greater than or equal to des(Asp1,Arg2)angiotensin II. Angiotensin III did not compete for binding to the site. High-pressure liquid chromatography analysis indicated that the ligand either in the incubation or bound to the site was stable at 15 degrees C, but there was very rapid and extensive degradation by the C6 glioma cells at 37 degrees C. It is concluded that the site exhibits unusual N-terminal specificity for angiotensin with nanomolar affinity for angiotensin II. If angiotensin III is an active ligand in the brain, the site may have a converting enzyme function. Alternatively, it may form the des-Asp derivatives of angiotensin for subsequent degradation by other enzymatic pathways. Either way, it is proposed that the site may modulate the brain-angiotensin system.

  10. Constrained H-Phe-Phe-NH2 analogues with high affinity to the substance P 1-7 binding site and with improved metabolic stability and cell permeability.

    Science.gov (United States)

    Fransson, Rebecca; Sköld, Christian; Kratz, Jadel M; Svensson, Richard; Artursson, Per; Nyberg, Fred; Hallberg, Mathias; Sandström, Anja

    2013-06-27

    We recently reported the discovery of H-Phe-Phe-NH2 as a small and high affinity ligand for the substance P 1-7 (SP(1-7), H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) specific binding site and its intriguing ability to reduce neuropathic pain. With the overall aim to develop stable and orally bioavailable SP(1-7) mimetics, the dipeptide was chosen as a lead compound. Herein the structure-activity relationship (SAR) of a set of modified H-Phe-Phe-NH2 analogues is presented together with their potential active uptake by PEPT1 transporter, intestinal permeability, and metabolic stability. Local constraints via peptide backbone methylation or preparation of cyclized analogues based on pyrrolidine were evaluated and were shown to significantly improve the in vitro pharmacokinetic properties. The SAR was rationalized by deriving a plausible binding pose for the high affinity ligands. Rigidification using a 3-phenylpyrrolidine moiety in the C-terminal of H-Phe-Phe-NH2 resulted in high affinity and improved intrinsic clearance and intestinal epithelial permeability. PMID:23735006

  11. Affinity Capillary Electrophoresis:Study of the Binding of HIV-1 gp41 with a Membrane Protein (P45) on the Human B Cell Line,Raji

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Affinity capillary electrophoresis has been used to study the interaction between a membrane protein (P45) isolated from the Human B cell line, Raji, and rsgp41. P45, rsgp41 and the complexes were well resolved. The entire separation was achieved in less than 3min. Formations of two kinds of stable P45-rsgp41 complexes were confirmed based on migration time comparison; the binding equilibrium was achieved as soon as two proteins were mixed. The results indicate that the interaction between P45 and rsgp41 is strong with a fast association rate and a slow dissociation rate, and there are at least two kinds of binding sites with different binding constants between P45 and rsgp41.

  12. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

    Science.gov (United States)

    Czikora, Agnes; Lundberg, Daniel J; Abramovitz, Adelle; Lewin, Nancy E; Kedei, Noemi; Peach, Megan L; Zhou, Xiaoling; Merritt, Raymond C; Craft, Elizabeth A; Braun, Derek C; Blumberg, Peter M

    2016-05-20

    The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. PMID:27022025

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

    OpenAIRE

    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 obtai...

  14. The increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: A potential for drug delivery system

    Science.gov (United States)

    Liu, Bing-Mi; Zhang, Jun; Hao, Ai-Jun; Xu, Liang; Wang, Dan; Ji, Hui; Sun, Shi-Jie; Chen, Bo-Qi; Liu, Bin

    2016-02-01

    The impacts of rutin and baicalin on the interaction of curcumin (CU) with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopies under imitated physiological conditions. The results showed that the fluorescence quenching of HSA by CU was a simultaneous static and dynamic quenching process, irrespective of the presence or absence of flavonoids. The binding constants between CU and HSA in the absence and presence of rutin and baicalin were 2.268 × 105 M- 1, 3.062 × 105 M- 1, and 3.271 × 105 M- 1, indicating that the binding affinity was increased in the case of two flavonoids. Furthermore, the binding distance determined according to Förster's theory was decreased in the presence of flavonoids. Combined with the fact that flavonoids and CU have the same binding site (site I), it can be concluded that they may simultaneously bind in different regions in site I, and formed a ternary complex of flavonoid-HSA-CU. Meanwhile, the results of fluorescence quenching, CD and three-dimensional fluorescence spectra revealed that flavonoids further strengthened the microenvironmental and conformational changes of HSA induced by CU binding. Therefore, it is possible to develop a novel complex involving CU, flavonoid and HSA for CU delivery. The work may provide some valuable information in terms of improving the poor bioavailabiliy of CU.

  15. Investigation of the Structure Requirement for 5-HT6 Binding Affinity of Arylsulfonyl Derivatives: A Computational Study

    Directory of Open Access Journals (Sweden)

    Hanqing Li

    2011-08-01

    Full Text Available 5-HT6 receptor has been implicated in a series of diseases including anxiety, depression, schizophrenia and cognitive dysfunctions. 5-HT6 ligands have been reported to play a significant role in the treatment for central nervous system (CNS diseases. Presently, a large series of 223 5-HT6 ligands were studied using a combinational method by 3D-QSAR, molecular docking and molecular dynamics calculations for further improvement of potency. The optimal 3D models exhibit satisfying statistical results with r2ncv, q2 values of 0.85 and 0.50 for CoMFA, 0.81 and 0.53 for CoMSIA, respectively. Their predictive powers were validated by external test set, showing r2pred of 0.71 and 0.76. The contour maps also provide a visual representation of contributions of steric, electrostatic, hydrophobic and hydrogen bond fields as well as the prospective binding models. In addition, the agreement between 3D-QSAR, molecular docking and molecular dynamics simulation proves the rationality of the developed models. These results, we hope, may be helpful in designing novel and potential 5-HT6 ligands.

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

    Czech Academy of Sciences Publication Activity Database

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

    2014-01-01

    Roč. 48, May (2014), s. 51-62. ISSN 0965-1748 Institutional support: RVO:61388963 Keywords : medfly * Ceratitis capitata * olfaction * odorant binding protein * pheromone binding protein * pheromone * binding studies * protein expression * electroantennography * GC-EAG * fluorescence displacement Subject RIV: CE - Biochemistry Impact factor: 3.450, year: 2014

  17. Variation in one residue associated with the metal ion-dependent adhesion site regulates αIIbβ3 integrin ligand binding affinity.

    Directory of Open Access Journals (Sweden)

    Joel Raborn

    Full Text Available The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala(252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala(252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.

  18. Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification.

    Science.gov (United States)

    von Rechenberg, Moritz; Blake, Brian Kelly; Ho, Yew-Seng J; Zhen, Yuejun; Chepanoske, Cindy Lou; Richardson, Bonnie E; Xu, Nafei; Kery, Vladimir

    2005-05-01

    The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors. PMID:15761956

  19. Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances

    International Nuclear Information System (INIS)

    The purpose of the present study was to determine whether ultraviolet light (UV) irradiation of amino acids produces compounds with affinity for the Ah receptor. Aqueous solutions of L-tryptophan were exposed to radiation from an unfiltered high-pressure mercury lamp. The photoproducts formed were solvent-extracted or concentrated on Sep-Pak C18 cartridges. The concentrated extracts or eluants were treated for their ability to compete with 3H-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding was assayed in liver cytosolic preparations from Sprague-Dawley rats using a technique based on hydroxylapatite separation. Photoproducts with receptor affinity were formed in a time-dependent manner. Histidine and tryptamine also gave products upon UV irradiation that competed with TCDD. Commercial tryptophan, at least aged, contained trace amounts of impurities with receptor affinity. Analysis by TLC and high-pressure liquid chromatography of the photo-products of tryptophan showed a minimum of three different binding compounds. Two of the products were studied in greater detail. One of them, showing UV absorbance and yellow fluorescence, gave a molecular ion (M+) of 284 and the other gave M+ 312 but showed little UV absorption and fluorescence. The concentration, based on mass spectrometry quantifications, of the two compounds that displaced more than 50% of TCDD was found to be extremely low, giving Kd values of 0.44 nM (M+ 312) and 0.07 nM (M+ 284). The existence of high affinity receptors for oxidized amino acids is postulated and their possible role in the proliferative cellular responses to TCDD and tryptophan is discussed briefly

  20. Silver nanoparticles for SERS-based ultrasensitive chemical detection in aqueous solutions: Role of binding affinity and surface oxidation in the detection limit

    Science.gov (United States)

    Erol, Melek

    Surface-enhanced Raman spectroscopy (SERS) in the presence of noble metal nanostructures holds significant promise for sensing and molecular fingerprinting down to single molecule level. This dissertation explores the effect of binding affinity and surface oxidation of Ag nanoparticles on SERS detection sensitivity of SO42-, CN-, SCN-, ClO4- and nitro-aromatic compounds in water. Specifically positively charged Ag nanoparticles (Ag [+]) were synthesized by UV-assisted reduction of silver nitrate using branched polyethyleneimine (BPEI) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) solutions. Both primary amino and amide groups on the surface of Ag [+] allowed strong binding affinity with anions, critical for sensitive SERS measurements. For substrates with immobilized Ag [+] (30 nanoparticles/mum2), SERS sensitivity increased in the order of SO42- physiological conditions due to steric hindrance from the branched architecture of adsorbed polymer chains. BPEI coated surfaces were also effective for suppression of smaller positively charged proteins such as lysozyme and ribonuclease A at pH 7 and 0.15 M NaCl and of negatively charged proteins such as BSA and fibrinogen at pH 7 and 0.75 M NaCl. Furthermore, using PEI-modified protein-repellent surfaces, selective binding of avidin was achieved to surface-bound Ag nanoparticles, thus providing a promising strategy for SERS-based bio-detection.

  1. Dot-ELISA affinity test: an easy, low-cost method to estimate binding activity of monoclonal antibodies

    Czech Academy of Sciences Publication Activity Database

    Svobodová, Z.; Jankovičová, B.; Horák, Daniel; Bílková, Z.

    2013-01-01

    Roč. 4, č. 3 (2013), 1000168_1-1000168_5. ISSN 2155-9872 R&D Projects: GA MŠk 7E12053; GA MŠk 7E09109 EU Projects: European Commission(XE) 246513 - NADINE; European Commission(XE) 228980 - CAMINEMS Institutional support: RVO:61389013 Keywords : dot-ELISA * affinity * monoclonal antibody Subject RIV: CD - Macromolecular Chemistry

  2. Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen.

    Science.gov (United States)

    Moon, Seung Kee; Park, So Ra; Park, Ami; Oh, Hyun Mi; Shin, Hyun Jung; Jeon, Eun Ju; Kim, Seiwhan; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je

    2016-03-31

    To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5. PMID:26743905

  3. High Affinity Binding of 3[H]-Cimetidine to a Heme-Containing Protein in Rat Brain

    OpenAIRE

    Stadel, Rebecca; Yang, Jun; Nalwalk, Julia W.; Phillips, James G.; Hough, Lindsay B.

    2007-01-01

    [3H]-Cimetidine (3HCIM) specifically binds to an unidentified site in the rat brain. Because recently-described ligands for this site have pharmacological activity, 3HCIM binding was presently characterized. 3HCIM binding was saturable, heat-labile, and distinct from the histamine H2 receptor. To test the hypothesis that 3HCIM binds to a cytochrome P450 (CYP), the effects of non-selective and isoform-selective CYP inhibitors were studied. The heme inhibitor KCN and the non-selective CYP inhib...

  4. Enhanced binding affinity, remarkable selectivity, and high capacity of CO 2 by dual functionalization of a rht-type metal-organic framework

    KAUST Repository

    Li, Baiyan

    2011-12-23

    Open and friendly: The smallest member of the rht-type metal-organic frameworks (MOFs, see picture) constructed by a hexacarboxylate ligand with a nitrogen-rich imino triazine backbone shows a significantly enhanced gas binding affinity relative to all other isoreticular rht-type MOFs. The high adsorption capacity and remarkable selectivity of CO 2 are attributed to the high density of open metal and Lewis basic sites in the framework. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Highly sensitive and selective ratiometric fluorescent copper sensors: Different binding affinities modulated by three separate side chains of naphthalimide

    Institute of Scientific and Technical Information of China (English)

    XU YuFang; LU Feng; XU ZhaoChao; CHENG TanYu; QIAN XuHong

    2009-01-01

    A series of compounds 1 --11 with different side chains of naphthalimide as fluorescent copper sensors were designed and synthesized. Compounds 1, 9, 10 and 11 presented a high selectivity to Cu2+ in a neutral aqueous environment. Here 1, 9 and 10 showed selectivity and affinity to Cu2+ with an association constant of about ~106. It gave somewhat response to Ag+, Co2+, Ni2+ and Fe2+ while 1 detected copper. 9 and 10 displayed better selectivity by changing their hydrophobic side chains to the hydrophilic ones on imide moieties. 11, with one flexible side chain, showed high selectivity and an association constant (Ka = 2.2 × 108), which were much higher than those of 1, 9 and 10. These results indicated that the selectivity and affinity could be improved by changing side chains of naphthalimide. That might provide a novel strategy or method for the development of fluorescent sensors.

  6. Low oxygen affinity in reptilian hemoglobin D: prediction of residue interactions in Geochelone carbonaria HbD by homology modeling.

    Science.gov (United States)

    Lutfullah, Ghosia; Khalil, Hilal Shahid; Amin, Farhat; Azhar, Noreen

    2008-04-01

    The homology model of hemoglobin D from Geochelone carbonaria, the red-footed tortoise was predicted using the 3D structure coordinates of Geochelone gigantea hemoglobin D as the template. The model was built using the program, MODELLER (8v1) and evaluated with PROCHECK and PROSA. The present study features an in-depth analysis of the 3D model and its conformational changes brought about with variations in its environment. These structural changes are correlated with its ability to adapt to different environmental constraints enabling the organism to better suit to its natural habitat. The model shows additional contacts between amino acid pairs of alpha-119 and beta-55, alpha-35 and beta-124, alpha-103 and beta-112, alpha-115 and beta-116, alpha-120 and beta-52, alpha-120 and beta-55, alpha-36 and beta-127 which are not found in human hemoglobin. It is predicted that these contacts may result in T-state stabilization thus lowering oxygen affinity. Furthermore, decrease in the interaction of phosphate heteroatoms with the amino acid residues of G. carbonaria Hb was also predicted in this study. PMID:18085430

  7. High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle

    International Nuclear Information System (INIS)

    Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species

  8. Analysis of drug-protein binding using on-line immunoextraction and high-performance affinity microcolumns: Studies with normal and glycated human serum albumin.

    Science.gov (United States)

    Matsuda, Ryan; Jobe, Donald; Beyersdorf, Jared; Hage, David S

    2015-10-16

    A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining drug-protein interactions with normal or modified proteins. Normal human serum albumin (HSA) and glycated HSA were used as model proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of protein at 0.05-0.10 mL/min and had a binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed proteins were tested for use in various approaches for drug binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these proteins for the drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this drug at Sudlow sites I and II of the adsorbed proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that drug-protein binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other drugs or proteins of interest in pharmaceutical studies or biomedical research. PMID:26381571

  9. Structure-function studies of DNA binding domain of response regulator KdpE reveals equal affinity interactions at DNA half-sites.

    Directory of Open Access Journals (Sweden)

    Anoop Narayanan

    Full Text Available Expression of KdpFABC, a K(+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABC(BS via the winged helix-turn-helix type DNA binding domain (KdpE(DBD. Exploration of E. coli KdpE(DBD and kdpFABC(BS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpE(DBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpE(DBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpE(DBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins.

  10. Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.

    Science.gov (United States)

    Rapali, Péter; Radnai, László; Süveges, Dániel; Harmat, Veronika; Tölgyesi, Ferenc; Wahlgren, Weixiao Y; Katona, Gergely; Nyitray, László; Pál, Gábor

    2011-01-01

    LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S](-4)K(-3)X(-2)[T/V/I](-1)Q(0)[T/V](1)[D/E](2). The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V(-5)S(-4)R(-3)G(-2)T(-1)Q(0)T(1)E(2) resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes

  11. Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.

    Directory of Open Access Journals (Sweden)

    Péter Rapali

    Full Text Available LC8 dynein light chain (DYNLL is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S](-4K(-3X(-2[T/V/I](-1Q(0[T/V](1[D/E](2. The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V(-5S(-4R(-3G(-2T(-1Q(0T(1E(2 resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes.

  12. Late pregnancy thyroid-binding globulin predicts perinatal depression.

    Science.gov (United States)

    Pedersen, Cort; Leserman, Jane; Garcia, Nacire; Stansbury, Melissa; Meltzer-Brody, Samantha; Johnson, Jacqueline

    2016-03-01

    Previously we found that late pregnancy total and free thyroxine (TT4, FT4) concentrations were negatively related to greater pre and/or postpartum depressive symptoms. In a much larger cohort, the current study examined whether these thyroid indices measured earlier in the third trimester (31-33 weeks) predict subsequent perinatal depression and anxiety ratings as well as syndromal depression. Thyroid-binding globulin (TBG) concentrations increase markedly during pregnancy and may be an index of sensitivity to elevated estrogen levels. TBG was examined in this study because prior findings suggest that postpartum depression is related to sensitivity to mood destabilization by elevated sex hormone concentrations during pregnancy. Our cohort was 199 euthyroid women recruited from a public health obstetrics clinic (63.8% Hispanic, 21.6% Black). After screening and blood draws for hormone measures at pregnancy weeks 31-33, subjects were evaluated during home visits at pregnancy weeks 35-36 as well as postpartum weeks 6 and 12. Evaluations included psychiatric interviews for current and life-time DSM-IV psychiatric history (M.I.N.I.-Plus), subject self-ratings and interviewer ratings for depression and anxiety (Edinburgh Postnatal Depression Scale, Montgomery-Ǻsberg Depression Rating Scale; Spielberger State-Trait Anxiety Inventory, Hamilton Anxiety Inventory), as well as a standardized interview to obtain life-time trauma history. Numerous covariates were included in all regression analyses. Trauma and major depression history were robustly significant predictors of depression and anxiety ratings over the study period when these variables were analyzed individually or in a combined model including FT4 or TBG (pdepression and anxiety ratings (pdepression history, were significant individual predictors of syndromal depression during the study period (pdepression history, FT4 and TBG generally were not significantly predictive of depression or anxiety ratings, and FT4

  13. Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor.

    OpenAIRE

    Kiefer, M C; Stephans, J C; Crawford, K; Okino, K.; Barr, P.J.

    1990-01-01

    Expression cloning of cDNAs encoding a basic fibroblast growth factor (FGF) binding protein confirms previous hypotheses that this molecule is a cell-surface heparan sulfate proteoglycan. A cDNA library constructed from a hamster kidney cell line rich in FGF receptor activity was transfected into a human lymphoblastoid cell line. Clones expressing functional basic FGF binding proteins at their surfaces were enriched by panning on plastic dishes coated with human basic FGF. The amino acid sequ...

  14. A Column Free Protein Purification Procedure using E. coli Single ‐ Stranded DNA Binding Protein (SSB) as an Affinity Tag

    OpenAIRE

    Soffe, Mark

    2014-01-01

    SSBs are DNA binding proteins that are essential components of cells and play key roles in DNA replication, repair, and recombination. Here we utilize two biochemical properties associated with the E. coli SSB protein to develop a novel procedure to purify proteins using a resin-free strategy to combat the largest bottleneck in biochemical research— obtaining uncontaminated, single proteins from the total cellular contents. 1. E. coli SSB binds to single stranded DNA (ssDNA) with extremely...

  15. Design, Synthesis, Binding and Docking-Based 3D-QSAR Studies of 2-Pyridylbenzimidazoles—A New Family of High Affinity CB1 Cannabinoid Ligands

    Directory of Open Access Journals (Sweden)

    Patricio Iturriaga-Vásquez

    2013-04-01

    Full Text Available A series of novel 2-pyridylbenzimidazole derivatives was rationally designed and synthesized based on our previous studies on benzimidazole 14, a CB1 agonist used as a template for optimization. In the present series, 21 compounds displayed high affinities with Ki values in the nanomolar range. JM-39 (compound 39 was the most active of the series (KiCB1 = 0.53 nM, while compounds 31 and 44 exhibited similar affinities to WIN 55212-2. CoMFA analysis was performed based on the biological data obtained and resulted in a statistically significant CoMFA model with high predictive value (q2 = 0.710, r2 = 0.998, r2pred = 0.823.

  16. LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation.

    Directory of Open Access Journals (Sweden)

    Clementine Menz

    Full Text Available Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2 belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor β (TGF-β, in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-β, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2 (Kd = 1.1 nM. Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like motifs (EGFs 9-14. This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.

  17. Biomimetic design of platelet adhesion inhibitors to block integrin α2β1-collagen interactions: I. Construction of an affinity binding model.

    Science.gov (United States)

    Zhang, Lin; Sun, Yan

    2014-04-29

    Platelet adhesion on a collagen surface through integrin α2β1 has been proven to be significant for the formation of arterial thrombus. However, the molecular determinants mediating the integrin-collagen complex remain unclear. In the present study, the dynamics of integrin-collagen binding and molecular interactions were investigated using molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis. Hydrophobic interaction is identified as the major driving force for the formation of the integrin-collagen complex. On the basis of the MD simulation and MM-PBSA results, an affinity binding model (ABM) of integrin for collagen is constructed; it is composed of five residues, including Y157, N154, S155, R288, and L220. The ABM has been proven to capture the major binding motif contributing 84.8% of the total binding free energy. On the basis of the ABM, we expect to establish a biomimetic design strategy of platelet adhesion inhibitors, which would be beneficial for the development of potent peptide-based drugs for thrombotic diseases. PMID:24697616

  18. Vsx2 controls eye organogenesis and retinal progenitor identity via homeodomain and non-homeodomain residues required for high affinity DNA binding.

    Directory of Open Access Journals (Sweden)

    Changjiang Zou

    2012-09-01

    Full Text Available The homeodomain and adjacent CVC domain in the visual system homeobox (VSX proteins are conserved from nematodes to humans. Humans with missense mutations in these regions of VSX2 have microphthalmia, suggesting both regions are critical for function. To assess this, we generated the corresponding mutations in mouse Vsx2. The homeodomain mutant protein lacked DNA binding activity and the knock-in mutant phenocopied the null mutant, ocular retardation J. The CVC mutant protein exhibited weakened DNA binding; and, although the corresponding knock-in allele was recessive, it unexpectedly caused the strongest phenotype, as indicated by severe microphthalmia and hyperpigmentation of the neural retina. This occurred through a cryptic transcriptional feedback loop involving the transcription factors Mitf and Otx1 and the Cdk inhibitor p27(Kip1. Our data suggest that the phenotypic severity of the CVC mutant depends on the weakened DNA binding activity elicited by the CVC mutation and a previously unknown protein interaction between Vsx2 and its regulatory target Mitf. Our data also suggest that an essential function of the CVC domain is to assist the homeodomain in high-affinity DNA binding, which is required for eye organogenesis and unhindered execution of the retinal progenitor program in mammals. Finally, the genetic and phenotypic behaviors of the CVC mutation suggest it has the characteristics of a recessive neomorph, a rare type of genetic allele.

  19. Predicting the binding free energy of the inclusion process of 2-hydroxypropyl-β-cyclodextrin and small molecules by means of the MM/3D-RISM method

    Science.gov (United States)

    Sugita, Masatake; Hirata, Fumio

    2016-09-01

    A protocol to calculate the binding free energy of a host–guest system is proposed based on the MM/3D-RISM method, taking cyclodextrin derivatives and their ligands as model systems. The protocol involves the procedure to identify the most probable binding mode (MPBM) of receptors and ligands by means of the umbrella sampling method. The binding free energies calculated by the MM/3D-RISM method for the complexes of the seven ligands with the MPBM of the cyclodextrin, and with the fluctuated structures around it, are in agreement with the corresponding experimental data in a semi-quantitative manner. It suggests that the protocol proposed here is promising for predicting the binding affinity of a small ligand to a relatively rigid receptor such as cyclodextrin.

  20. Predicting the binding free energy of the inclusion process of 2-hydroxypropyl-β-cyclodextrin and small molecules by means of the MM/3D-RISM method.

    Science.gov (United States)

    Sugita, Masatake; Hirata, Fumio

    2016-09-28

    A protocol to calculate the binding free energy of a host-guest system is proposed based on the MM/3D-RISM method, taking cyclodextrin derivatives and their ligands as model systems. The protocol involves the procedure to identify the most probable binding mode (MPBM) of receptors and ligands by means of the umbrella sampling method. The binding free energies calculated by the MM/3D-RISM method for the complexes of the seven ligands with the MPBM of the cyclodextrin, and with the fluctuated structures around it, are in agreement with the corresponding experimental data in a semi-quantitative manner. It suggests that the protocol proposed here is promising for predicting the binding affinity of a small ligand to a relatively rigid receptor such as cyclodextrin. PMID:27452185

  1. Computational Prediction of Heme-Binding Residues by Exploiting Residue Interaction Network

    OpenAIRE

    Rong Liu; Jianjun Hu

    2011-01-01

    Computational identification of heme-binding residues is beneficial for predicting and designing novel heme proteins. Here we proposed a novel method for heme-binding residue prediction by exploiting topological properties of these residues in the residue interaction networks derived from three-dimensional structures. Comprehensive analysis showed that key residues located in heme-binding regions are generally associated with the nodes with higher degree, closeness and betweenness, but lower ...

  2. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.

    Science.gov (United States)

    Torres, Oscar B; Antoline, Joshua F G; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2016-02-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not

  3. Sugar Binding Residue Affects Apparent Na+ Affinity and Transport Stoichiometry in Mouse Sodium/Glucose Cotransporter Type 3B*

    OpenAIRE

    Díez-Sampedro, Ana; Barcelona, Stephanie

    2010-01-01

    SGLT1 is a sodium/glucose cotransporter that moves two Na+ ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting t...

  4. Extraction of fibre network architecture by X-ray tomography and prediction of elastic properties using an affine analytical model

    International Nuclear Information System (INIS)

    This paper proposes a method for extracting reliable architectural characteristics from complex porous structures using micro-computed tomography (μCT) images. The work focuses on a highly porous material composed of a network of fibres bonded together. The segmentation process, allowing separation of the fibres from the remainder of the image, is the most critical step in constructing an accurate representation of the network architecture. Segmentation methods, based on local and global thresholding, were investigated and evaluated by a quantitative comparison of the architectural parameters they yielded, such as the fibre orientation and segment length (sections between joints) distributions and the number of inter-fibre crossings. To improve segmentation accuracy, a deconvolution algorithm was proposed to restore the original images. The efficacy of the proposed method was verified by comparing μCT network architectural characteristics with those obtained using high resolution CT scans (nanoCT). The results indicate that this approach resolves the architecture of these complex networks and produces results approaching the quality of nanoCT scans. The extracted architectural parameters were used in conjunction with an affine analytical model to predict the axial and transverse stiffnesses of the fibre network. Transverse stiffness predictions were compared with experimentally measured values obtained by vibration testing.

  5. Extraction of fibre network architecture by X-ray tomography and prediction of elastic properties using an affine analytical model

    Energy Technology Data Exchange (ETDEWEB)

    Tsarouchas, D. [Department of Engineering, University of Cambridge, Trumpington Street, Cambridge CB2 1PZ (United Kingdom); Markaki, A.E., E-mail: am253@cam.ac.uk [Department of Engineering, University of Cambridge, Trumpington Street, Cambridge CB2 1PZ (United Kingdom)

    2011-10-15

    This paper proposes a method for extracting reliable architectural characteristics from complex porous structures using micro-computed tomography ({mu}CT) images. The work focuses on a highly porous material composed of a network of fibres bonded together. The segmentation process, allowing separation of the fibres from the remainder of the image, is the most critical step in constructing an accurate representation of the network architecture. Segmentation methods, based on local and global thresholding, were investigated and evaluated by a quantitative comparison of the architectural parameters they yielded, such as the fibre orientation and segment length (sections between joints) distributions and the number of inter-fibre crossings. To improve segmentation accuracy, a deconvolution algorithm was proposed to restore the original images. The efficacy of the proposed method was verified by comparing {mu}CT network architectural characteristics with those obtained using high resolution CT scans (nanoCT). The results indicate that this approach resolves the architecture of these complex networks and produces results approaching the quality of nanoCT scans. The extracted architectural parameters were used in conjunction with an affine analytical model to predict the axial and transverse stiffnesses of the fibre network. Transverse stiffness predictions were compared with experimentally measured values obtained by vibration testing.

  6. Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity

    International Nuclear Information System (INIS)

    Tumor necrosis factor (TNF) is a proteinaceous toxin shed by stimulated myeloid cells. Murine TNF was radioiodinated to a specific activity of 1 mCi/nmol (1 Ci = 37 GBq) of monomer. 125I-labeled TNF (125-TNF) retained complete cytotoxic activity and it was immunochemically identical to the native toxin in a quantitative immunoprecipitation assay. It could be shown by competition binding that 125I-TNF bound to intact L929 cells with a specificity equal to that of native toxin. The conditions of time, temperature, and concentration involved in equilibrium specific binding to intact cells were studied in detail. J774.1 cells, the source of the toxin, demonstrated similar binding but were not sensitive to 125I-TNF cytotoxicity. Normal lymphoid organ cell suspensions and two human tumorigenic cell lines were not sensitive and failed to demonstrate specific binding. 125I-TNF, covalently cross-linked to its receptor on sensitive L-M cells with disuccinimidyl suberate, was isolated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. Two specific bands were identified. The presence of the binding site appears to be necessary but not sufficient to explain the sensitivity of cells to the cytotoxic action of TNF

  7. Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach

    DEFF Research Database (Denmark)

    Jüse, Ulrike; Arntzen, Magnus; Højrup, Peter;

    2011-01-01

    Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount...... of HLA molecules, giving a selective force in the binding. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides...... bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the...

  8. Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

    International Nuclear Information System (INIS)

    N-(4-Azidosalicyl) galactosamine (GalNASA), a photoactivatable, radioiodinatable analog of N-acetylgalactosamine (GalNAc), has been prepared and characterized. The authors have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a K/sub i,app/ of 800 μM, comparable to that of GalNAc. The K/sub i,app/ of GalNASA decreased to 40 μm upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl) ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with 125I-GalNASA was entirely dependent upon ultraviolet light. A portion of labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethyl-enediaminetetraacetic acid. The carbohydrate-sensitive fraction of discoidin I photolabeling with 125I-GalNASA exhibited a K/sub d/ of 15-40 μM, in agreement with the K/sub i,app/ of prephotolyzed GalNASA observed in the carbohydrate binding assay. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc. This indicated that the location of carbohydrate-sensitive labeling within the structure of discoidin I was restricted. One particular tryptic fragment, Tr1, was examined in detail. These data suggest that Tr1 is derived from the carbohydrate binding site of discoidin I

  9. In Silico Characterization of the Binding Affinity of Dendrimers to Penicillin-Binding Proteins (PBPs): Can PBPs be Potential Targets for Antibacterial Dendrimers?

    Science.gov (United States)

    Ahmed, Shaimaa; Vepuri, Suresh B; Ramesh, Muthusamy; Kalhapure, Rahul; Suleman, Nadia; Govender, Thirumala

    2016-04-01

    We have shown that novel silver salts of poly (propyl ether) imine (PETIM) dendron and dendrimers developed in our group exhibit preferential antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus. This led us to examine whether molecular modeling methods could be used to identify the key structural design principles for a bioactive lead molecule, explore the mechanism of binding with biological targets, and explain their preferential antibacterial activity. The current article reports the conformational landscape as well as mechanism of binding of generation 1 PETIM dendron and dendrimers to penicillin-binding proteins (PBPs) in order to understand the antibacterial activity profiles of their silver salts. Molecular dynamics at different simulation protocols and conformational analysis were performed to elaborate on the conformational features of the studied dendrimers, as well as to create the initial structure for further binding studies. The results showed that for all compounds, there were no significant conformational changes due to variation in simulation conditions. Molecular docking calculations were performed to investigate the binding theme between the studied dendrimers and PBPs. Interestingly, in significant accordance with the experimental data, dendron and dendrimer with aliphatic cores were found to show higher activity against S. aureus than the dendrimer with an aromatic core. The latter showed higher activity against MRSA. The findings from this computational and molecular modeling report together with the experimental results serve as a road map toward designing more potent antibacterial dendrimers against resistant bacterial strains. PMID:26743758

  10. Biochemistry of terminal deoxynucleotidyltransferase. Affinity labeling and identification of the deoxynucleoside triphosphate binding domain of terminal deoxynucleotidyltransferase

    International Nuclear Information System (INIS)

    Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites, we have labeled calf terminal deoxynucleotidyltransferase (TdT) with [32P]dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, requiring concentrations consistent with their kinetic constants. Tryptic peptide mapping of the [32P]dTTP-labeled enzyme showed the presence of a single radioactive peptide fraction that contained the site of dTTP cross-linking. The amino acid composition and sequence analysis of the radioactive peptide fraction revealed it to contain two tryptic peptides, spanning residues 221-231 and 234-249. Since these two peptides were covalently linked to dTTP, the region encompassed by them constitutes a substrate binding domain in TdT. Further proteolytic digestion of the tryptic peptide-dTTP complex, using V8 protease, yielded a smaller peptide, and its analysis narrowed the substrate binding domain to 14 amino acids corresponding to residues 224-237 in the primary amino acid sequence of TdT. Furthermore, 2 cysteine residues, Cys-227 and Cys-234, within this domain were found to be involved in the cross-linking of dTTP, suggesting their participation in the process of substrate binding in TdT

  11. Characterization of SynCAM surface trafficking using a SynCAM derived ligand with high homophilic binding affinity

    International Nuclear Information System (INIS)

    In order to better probe SynCAM function in neurons, we produced a fusion protein between the extracellular domain of SynCAM1 and the constant fragment of human IgG (SynCAM-Fc). Whether in soluble form or immobilized on latex microspheres, the chimera bound specifically to the surface of hippocampal neurons and recruited endogenous SynCAM molecules. SynCAM-Fc was also used in combination with Quantum Dots to follow the mobility of transfected SynCAM receptors at the neuronal surface. Both immobile and highly mobile SynCAM were found. Thus, SynCAM-Fc behaves as a high affinity ligand that can be used to study the function of SynCAM at the neuronal membrane

  12. A new real-time method for investigation of affinity properties and binding kinetics of magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Orlov, Alexey V. [Prokhorov General Physics Institute, Russian Academy of Sciences, 38 Vavilov St, 119991 Moscow (Russian Federation); Nikitin, Maxim P. [Prokhorov General Physics Institute, Russian Academy of Sciences, 38 Vavilov St, 119991 Moscow (Russian Federation); Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology, 9 Institutskii per., Dolgoprudny, Moscow Region 141700 (Russian Federation); Bragina, Vera A.; Znoyko, Sergey L.; Zaikina, Marina N.; Ksenevich, Tatiana I.; Gorshkov, Boris G. [Prokhorov General Physics Institute, Russian Academy of Sciences, 38 Vavilov St, 119991 Moscow (Russian Federation); Nikitin, Petr I., E-mail: nikitin@kapella.gpi.ru [Prokhorov General Physics Institute, Russian Academy of Sciences, 38 Vavilov St, 119991 Moscow (Russian Federation); National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 31 Kashirskoe shosse, 115409 Moscow (Russian Federation)

    2015-04-15

    A method for quantitative investigation of affinity constants of receptors immobilized on magnetic nanoparticles (MP) is developed based on spectral correlation interferometry (SCI). The SCI records with a picometer resolution the thickness changes of a layer of molecules or nanoparticles due to a biochemical reaction on a cover slip, averaged over the sensing area. The method is compatible with other types of sensing surfaces employed in biosensing. The measured values of kinetic association constants of magnetic nanoparticles are 4 orders of magnitude higher than those of molecular antibody association with antigen. The developed method also suggests highly sensitive detection of antigens in a wide dynamic range. The limit of detection of 92 pg/ml has been demonstrated for prostate-specific antigen (PSA) with 50-nm MP employed as labels, which produce 3-order amplification of the SCI signals. The calibration curve features high sensitivity (slope) of 3-fold signal raise per 10-fold increase of PSA concentration within 4-order dynamic range, which is an attractive compromise for precise quantitative and highly sensitive immunoassay. The proposed biosensing technique offers inexpensive disposable sensor chips of cover slips and represents an economically sound alternative to traditional immunoassays for disease diagnostics, detection of pathogens in food and environmental monitoring. - Highlights: • Method for study of affinity constants of magnetic nanoparticles with receptors is proposed. • Association constants of such particles are 4 orders higher than for biomolecules. • Method is compatible with widely used biosensor surfaces and affordable consumables. • It has high sensitivity: 3-fold signal increasing per 10-fold of PSA concentration. • Limit of detection for PSA is 92 pg/ml, dynamic range – 4 orders of concentration.

  13. Palmitic acid analogs exhibit nanomolar binding affinity for the HIV-1 CD4 receptor and nanomolar inhibition of gp120-to-CD4 fusion.

    Directory of Open Access Journals (Sweden)

    Elena E Paskaleva

    Full Text Available BACKGROUND: We recently reported that palmitic acid (PA is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (K(d and gp120-to-CD4 inhibition constants (K(i. The PA analogs were selected to satisfy Lipinski's rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity. PRINCIPAL FINDINGS: PA analog 2-bromopalmitate (2-BP was most efficacious with K(d approximately 74 nM and K(i approximately 122 nM, ascorbyl palmitate (6-AP exhibited slightly higher K(d approximately 140 nM and K(i approximately 354 nM, and sucrose palmitate (SP was least efficacious binding to CD4 with K(d approximately 364 nM and inhibiting gp120-to-CD4 binding with K(i approximately 1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry. SIGNIFICANCE: Considering observed differences between K(i and K(d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry.

  14. Positron emission tomography displacement sensitivity: predicting binding potential change for positron emission tomography tracers based on their kinetic characteristics.

    Science.gov (United States)

    Morris, Evan D; Yoder, Karmen K

    2007-03-01

    There is great interest in positron emission tomography (PET) as a noninvasive assay of fluctuations in synaptic neurotransmitter levels, but questions remain regarding the optimal choice of tracer for such a task. A mathematical method is proposed for predicting the utility of any PET tracer as a detector of changes in the concentration of an endogenous competitor via displacement of the tracer (a.k.a., its 'vulnerability' to competition). The method is based on earlier theoretical work by Endres and Carson and by the authors. A tracer-specific predictor, the PET Displacement Sensitivity (PDS), is calculated from compartmental model simulations of the uptake and retention of dopaminergic radiotracers in the presence of transient elevations of dopamine (DA). The PDS predicts the change in binding potential (DeltaBP) for a given change in receptor occupancy because of binding by the endogenous competitor. Simulations were performed using estimates of tracer kinetic parameters derived from the literature. For D(2)/D(3) tracers, the calculated PDS indices suggest a rank order for sensitivity to displacement by DA as follows: raclopride (highest sensitivity), followed by fallypride, FESP, FLB, NMSP, and epidepride (lowest). Although the PDS takes into account the affinity constant for the tracer at the binding site, its predictive value cannot be matched by either a single equilibrium constant, or by any one rate constant of the model. Values for DeltaBP have been derived from published studies that employed comparable displacement paradigms with amphetamine and a D(2)/D(3) tracer. The values are in good agreement with the PDS-predicted rank order of sensitivity to displacement. PMID:16788713

  15. An explicitly solvated full atomistic model of the cardiac thin filament and application on the calcium binding affinity effects from familial hypertrophic cardiomyopathy linked mutations

    Science.gov (United States)

    Williams, Michael; Schwartz, Steven

    2015-03-01

    The previous version of our cardiac thin filament (CTF) model consisted of the troponin complex (cTn), two coiled-coil dimers of tropomyosin (Tm), and 29 actin units. We now present the newest revision of the model to include explicit solvation. The model was developed to continue our study of genetic mutations in the CTF proteins which are linked to familial hypertrophic cardiomyopathies. Binding of calcium to the cTnC subunit causes subtle conformational changes to propagate through the cTnC to the cTnI subunit which then detaches from actin. Conformational changes propagate through to the cTnT subunit, which allows Tm to move into the open position along actin, leading to muscle contraction. Calcium disassociation allows for the reverse to occur, which results in muscle relaxation. The inclusion of explicit TIP3 water solvation allows for the model to get better individual local solvent to protein interactions; which are important when observing the N-lobe calcium binding pocket of the cTnC. We are able to compare in silica and in vitro experimental results to better understand the physiological effects from mutants, such as the R92L/W and F110V/I of the cTnT, on the calcium binding affinity compared to the wild type.

  16. Role of hydrophobic mutations on the binding affinity and stability of blood coagulation factor VIIIa: a computational molecular dynamics and free-energy analysis.

    Science.gov (United States)

    Venkateswarlu, Divi

    2014-07-18

    Factor VIIIa is a non-covalently bound hetero-trimer among A1, A2 and A3-C1-C2 domains and an essential co-factor for factor IXa enzyme during proteolytic activation of factor X zymogen. The relatively weak interactions between A2 and the interface A1/A3 domains dampen the functional stability of FVIIIa in plasma and results in rapid degradation. We studied the mutational effect of three charged residues (Asp519, Glu665 and Asp666) to several hydrophobic residues by molecular dynamics simulations. Analysis of the binding free energy by MM-PBSA and MM-GBSA methods shows that the mutation of Asp519 and Glu665 residues to either Val or Ala enhance the A2 domain binding affinity in agreement with the experimental site-specific mutagenesis data. Mutation of Asp666 to Val, Tyr, Met and Phe showed largest improvement in the A2-domain binding among the eight hydrophobic mutants studied. Our studies suggest that the enrichment of hydrophobic interactions in the buried surface regions of A2 domain plays crucial role in improving the overall stability of FVIIIa. PMID:24952158

  17. Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS

    Science.gov (United States)

    Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G.; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J.; Filizola, Marta; Springer, Timothy A.; Coller, Barry S.

    2012-01-01

    An integrin found on platelets, αIIbβ3 mediates platelet aggregation, and αIIbβ3 antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the β subunit metal ion–dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the αIIb subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the β3 subunits. They induce conformational changes in the β3 subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of αIIbβ3 (RUC-1) that binds exclusively to the αIIb subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β3 as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2–IIbβ3 headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb the way RUC-1 does, but in addition, it binds to the β3 MIDAS residue glutamic acid 220, thus displacing Mg2+ from the MIDAS. When the Mg2+ concentration was increased to 20 mM, however, Mg2+ was identified in the MIDAS and RUC-2 was absent. RUC-2′s ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg2+ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other αIIbβ3 antagonists and may offer advantages as a therapeutic agent. PMID:22422993

  18. A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

    KAUST Repository

    Chen, Peng

    2015-12-03

    Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

  19. Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Sheppard, P O; Jensen, L B;

    2001-01-01

    The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a...... "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and...... the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for...

  20. High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin

    OpenAIRE

    Saris, Jasper; Derkx, Frans; Bruin, René; Dekkers, Dick; Lamers, Jos; Saxena, Pramod Ranjan; Schalekamp, Maarten; Danser, Jan

    2001-01-01

    textabstractMannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell ...

  1. Automated Solution-Phase Synthesis of Insect Glycans to Probe the Binding Affinity of Pea Enation Mosaic Virus.

    Science.gov (United States)

    Tang, Shu-Lun; Linz, Lucas B; Bonning, Bryony C; Pohl, Nicola L B

    2015-11-01

    Pea enation mosaic virus (PEMV)--a plant RNA virus transmitted exclusively by aphids--causes disease in multiple food crops. However, the aphid-virus interactions required for disease transmission are poorly understood. For virus transmission, PEMV binds to a heavily glycosylated receptor aminopeptidase N in the pea aphid gut and is transcytosed across the gut epithelium into the aphid body cavity prior to release in saliva as the aphid feeds. To investigate the role of glycans in PEMV-aphid interactions and explore the possibility of viral control through blocking a glycan interaction, we synthesized insect N-glycan terminal trimannosides by automated solution-phase synthesis. The route features a mannose building block with C-5 ester enforcing a β-linkage, which also provides a site for subsequent chain extension. The resulting insect N-glycan terminal trimannosides with fluorous tags were used in a fluorous microarray to analyze binding with fluorescein isothiocyanate-labeled PEMV; however, no specific binding between the insect glycan and PEMV was detected. To confirm these microarray results, we removed the fluorous tag from the trimannosides for isothermal titration calorimetry studies with unlabeled PEMV. The ITC studies confirmed the microarray results and suggested that this particular glycan-PEMV interaction is not involved in virus uptake and transport through the aphid. PMID:26457763

  2. "Assessment of human AT1 Binding Affinity of Some Novel 2-alkylthio-1-[4-(N-α-ethoxycarbonyl-nzylaminobenzyl-5-hydroxymethylimidazoles "

    Directory of Open Access Journals (Sweden)

    Setareh Badakhshannoory

    2004-06-01

    Full Text Available Antagonists of various components of the renin-angiotensin system have been the subject of many studies for the control of blood pressure. Compounds with a phenoxyphenylacetic acid moiety that mimic the structure of losartan which is a powerful competitive antagonist of angiotensin receptor, have shown to be effective. In this study, the affinity of some 2-alkylthio-1-[4-(N-α-ethoxycarbonylbenzylaminobenzyl]-5-hydroxymethyl imidazoles for the human AT1 receptor was assessed in a radioligand binding assay. It was found that an alkyl chain of appropriate length would be most suitable if situated on the imidazole ring. Furthermore, variations of the lower phenyl rings demonstrated that introduction of a methyl group in this position will account for the most desired effect.

  3. Molecular basis for the affinity and specificity in the binding of five-membered iminocyclitols with glycosidases: an experimental and theoretical synergy.

    Science.gov (United States)

    Shandilya, Ashutosh; Ganesan, Muthupandian; Parveen, Firdaus; Ramesh, Namakkal G; Jayaram, B

    2016-06-24

    An unusual substituent dictated complementarity in the inhibition of amino-modified five-membered iminocyclitols toward various glycosidases was reported by us. These intriguing results encouraged us to seek a molecular level explanation for the observation that may facilitate the design of specific iminocyclitol inhibitors against glycosidases of choice. We present here a detailed theoretical account that is substantiated with some new experimental investigations on the molecular origins of the differential affinities of iminocyclitols with various glycosidases. The studies involve docking/scoring, molecular dynamics simulations followed by syntheses of a few novel five-membered iminocyclitols and their in vitro binding assays. Directional hydrogen bonds and snug fit of the ligands are implicated as contributory to the observed selectivities. The observed synergy between the computations and experiment is likely to spur further research in the design of novel iminocyclitols with specific inhibitory activities. PMID:27085905

  4. LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

    Science.gov (United States)

    Fliegmann, Judith; Jauneau, Alain; Pichereaux, Carole; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Burlet-Schiltz, Odile; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-01

    LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves. PMID:27129432

  5. Neurotensin high affinity binding sites and endopeptidase 24. 11 are present respectively in the meningothelial and in the fibroblastic components of human meningiomas

    Energy Technology Data Exchange (ETDEWEB)

    Mailleux, P.; Przedborski, S.; Beaumont, A.; Verslijpe, M.; Depierreux, M.; Levivier, M.; Kitabgi, P.; Roques, B.P.; Vanderhaeghen, J.J. (Brugmann Hospital, Bruxelles (Belgium))

    1990-11-01

    The presence of neurotensin receptors and endopeptidase 24.11 (E-24.11) in 16 human meningioma specimens, obtained at surgery, was assessed by measuring the binding of {sup 125}I-(tyrosyl3)neurotensin(1-13) ({sup 125}I-NT) and the inhibitor {sup 3}H-N(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-(oxopropyl)glycine ({sup 3}H-HACBO-Gly), for the receptor and enzyme, respectively. E-24.11 activity was also measured. Autoradiography, on the 16 meningiomas, showed that specific {sup 125}I-NT labeling (nonspecific labeling was assessed in the presence of excess NT) was exclusively located in the meningothelial regions. In contrast, specific {sup 3}H-HACBO-Gly labeling (nonspecific labeling was assessed in the presence of an excess of the E-24.11 inhibitor thiorphan) was exclusively found in fibroblastic regions. No specific labeling of either ligand was found on collagen or blood vessels. In vitro binding assays were performed on membranes of 10 of the 16 meningiomas. In the 4 meningiomas rich in meningothelial cells, {sup 125}I-NT specifically bound to one population of sites with Bmax ranging from 57 to 405 fmol/mg protein and Kd around 0.3 nM. These sites share common properties with the brain NT receptor, since the carboxy terminal acetyl NT(8-13) fragment bound to the same sites but with a higher affinity. The carboxy terminal analogue of NT, neuromedin N, also bound to the same sites with a 10-fold lower affinity and the sites were bradykinin and levocabastine insensitive. In the 4 meningiomas rich in fibroblastic cells, {sup 3}H-HACBO-Gly specifically bound to one population of sites with Bmax ranging from 251 to 739 fmol/mg protein and Kd around 2.8 nM.

  6. Exploring the Origin of Differential Binding Affinities of Human Tubulin Isotypes αβII, αβIII and αβIV for DAMA-Colchicine Using Homology Modelling, Molecular Docking and Molecular Dynamics Simulations.

    Science.gov (United States)

    Kumbhar, Bajarang Vasant; Borogaon, Anubhaw; Panda, Dulal; Kunwar, Ambarish

    2016-01-01

    Tubulin isotypes are found to play an important role in regulating microtubule dynamics. The isotype composition is also thought to contribute in the development of drug resistance as tubulin isotypes show differential binding affinities for various anti-cancer agents. Tubulin isotypes αβII, αβIII and αβIV show differential binding affinity for colchicine. However, the origin of differential binding affinity is not well understood at the molecular level. Here, we investigate the origin of differential binding affinity of a colchicine analogue N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchicine) for human αβII, αβIII and αβIV isotypes, employing sequence analysis, homology modeling, molecular docking, molecular dynamics simulation and MM-GBSA binding free energy calculations. The sequence analysis study shows that the residue compositions are different in the colchicine binding pocket of αβII and αβIII, whereas no such difference is present in αβIV tubulin isotypes. Further, the molecular docking and molecular dynamics simulations results show that residue differences present at the colchicine binding pocket weaken the bonding interactions and the correct binding of DAMA-colchicine at the interface of αβII and αβIII tubulin isotypes. Post molecular dynamics simulation analysis suggests that these residue variations affect the structure and dynamics of αβII and αβIII tubulin isotypes, which in turn affect the binding of DAMA-colchicine. Further, the binding free-energy calculation shows that αβIV tubulin isotype has the highest binding free-energy and αβIII has the lowest binding free-energy for DAMA-colchicine. The order of binding free-energy for DAMA-colchicine is αβIV ≃ αβII > αβIII. Thus, our computational approaches provide an insight into the effect of residue variations on differential binding of αβII, αβIII and αβIV tubulin isotypes with DAMA-colchicine and may help to design new analogues with higher

  7. Synthesis and Evaluation of In Vitro DNA/Protein Binding Affinity, Antimicrobial, Antioxidant and Antitumor Activity of Mononuclear Ru(II) Mixed Polypyridyl Complexes.

    Science.gov (United States)

    Putta, Venkat Reddy; Chintakuntla, Nagamani; Mallepally, Rajender Reddy; Avudoddi, Srishailam; K, Nagasuryaprasad; Nancherla, Deepika; V V N, Yaswanth; R S, Prakasham; Surya, Satyanarayana Singh; Sirasani, Satyanarayana

    2016-01-01

    The four novel Ru(II) complexes [Ru(phen)2MAFIP](2+) (1) [MAFIP = 2-(5-(methylacetate)furan-2-yl)-1 H-imidazo[4,5-f] [1, 10]phenanthroline, phen = 1,10-Phenanthroline], [Ru(bpy)2MAFIP](2+) (2) (bpy = 2,2'-bipyridine) and [Ru(dmb)2MAFIP](2+) (3) (dmb = 4,4'-dimethyl-2,2'-bipyridine) and [Ru(hdpa)2MAFIP](2+) (4) (hdpa = 2,2-dipyridylamine) have been synthesized and fully characterized via elemental analysis, NMR spectroscopy, EI-MS and FT-IR spectroscopy. In addition, the DNA-binding behaviors of the complexes 1-4 with calf thymus DNA were investigated by UV-Vis absorption, fluorescence studies and viscosity measurement. The DNA-binding experiments showed that the complexes 1-4 interact with CT-DNA through an intercalative mode. BSA protein binding affinity of synthesized complexes was determined by UV/Vis absorption and fluorescence emission titrations. The binding affinity of ruthenium complexes was supported by molecular docking. The photoactivated cleavage of plasmid pBR322 DNA by ruthenium complexes 1-4 was investigated. All the synthesized compounds were tested for antimicrobial activity by using three Gram-negative (Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa) and three Gram-positive (Micrococcus luteus, Bacillus subtilis and Bacillus megaterium) organisms, these results indicated that complex 3 was more activity compared to other complexes against all tested microbial strains while moderate antimicrobial activity profile was noticed for complex 4. The antioxidant activity experiments show that the complexes exhibit moderate antioxidant activity. The cytotoxicity of synthesized complexes on HeLa cell lines has been examined by MTT assay. The apoptosis assay was carried out with Acridine Orange (AO) staining methods and the results indicate that complexes can induce the apoptosis of HeLa cells. The cell cycle arrest investigated by flow cytometry and these results indicate that complexes 1-4 induce the cell cycle arrest at G0/G1

  8. MHC class I epitope binding prediction trained on small data sets

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Nielsen, Morten; Lamberth, K.; Worning, Peder; Sylvester-Hvid, C.; Buus, S.; Brunak, Søren; Lund, Ole

    predicting peptides binding to specific MHC class I alleles. The method combines advanced automatic scoring matrix generation with empirical position specific differential anchor weighting. The method leads to predictions with a comparable or higher accuracy than other established prediction servers, even in...... situations where only very limited data are available for training....

  9. An Overview of the Prediction of Protein DNA-Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

    Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  10. Dissecting the Relation between a nuclear receptor and GATA: binding affinity studies of thyroid hormone receptor and GATA2 on TSHβ promoter.

    Directory of Open Access Journals (Sweden)

    Ana Carolina Migliorini Figueira

    Full Text Available BACKGROUND: Much is known about how genes regulated by nuclear receptors (NRs are switched on in the presence of a ligand. However, the molecular mechanism for gene down-regulation by liganded NRs remains a conundrum. The interaction between two zinc-finger transcription factors, Nuclear Receptor and GATA, was described almost a decade ago as a strategy adopted by the cell to up- or down-regulate gene expression. More recently, cell-based assays have shown that the Zn-finger region of GATA2 (GATA2-Zf has an important role in down-regulation of the thyrotropin gene (TSHβ by liganded thyroid hormone receptor (TR. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to better understand the mechanism that drives TSHβ down-regulation by a liganded TR and GATA2, we have carried out equilibrium binding assays using fluorescence anisotropy to study the interaction of recombinant TR and GATA2-Zf with regulatory elements present in the TSHβ promoter. Surprisingly, we observed that ligand (T3 weakens TR binding to a negative regulatory element (NRE present in the TSHβ promoter. We also show that TR may interact with GATA2-Zf in the absence of ligand, but T3 is crucial for increasing the affinity of this complex for different GATA response elements (GATA-REs. Importantly, these results indicate that TR complex formation enhances DNA binding of the TR-GATA2 in a ligand-dependent manner. CONCLUSIONS: Our findings extend previous results obtained in vivo, further improving our understanding of how liganded nuclear receptors down-regulate gene transcription, with the cooperative binding of transcription factors to DNA forming the core of this process.

  11. Correction: Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding

    OpenAIRE

    Banerjee, Sugato; Evanson, Janel; Harris, Erik; Lowe, Stephen L; Speth, Robert C.; Thomasson, Kathryn A; Porter, James E.

    2006-01-01

    This is a correction article. After publication of this work [1], we became aware of the fact that Robert C. Speth was not included as an author. Dr. Speth put a considerable amount of time and effort into developing and preparing the radiopeptide used to carry out the radioligand binding studies reported in this manuscript and therefore should have originally been included as an author. We apologize to Dr. Speth for any inconvenience that this oversight might have caused and thank him for hi...

  12. The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

    OpenAIRE

    van Nuland Nico AJ; Lopez-Mayorga Obdulio; Eshuis Henk; AB Eiso; Casares Salvador; Conejero-Lara Francisco

    2007-01-01

    Abstract Background SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 t...

  13. The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

    OpenAIRE

    Casares, Salvador; AB Eiso; Eshuis, Henk; Lopez-Mayorga, Obdulio; van Nuland, Nico AJ; Conejero-Lara, Francisco

    2007-01-01

    Background SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lowe...

  14. Striatal binding of 2-amino-6,7-(/sup 3/H)dihydroxy-1,2,3,4-tetrahydronaphthalene to two dopaminergic sites distinguished by their low and high affinity for neuroleptics

    Energy Technology Data Exchange (ETDEWEB)

    List, S.J.; Wreggett, K.A.; Seeman, P.

    1982-07-01

    In order to develop more selective methods for labeling brain dopamine receptors, this study describes in detail the properties of 2-amino-6,7,-(/sup 3/H)dihydroxy-1,2,3,4,-tetrahydronaphthalene ((/sup 3/H) ADTN) binding to dopaminergic sites in rat, calf, and human brain. (/sup 3/H)ADTN labeled two distinct types of dopaminergic binding sites in the brain striatum of the rat, calf, and human. Very low concentrations of dopamine and dopaminergic catecholamines (with IC50 values of 1 to 10 nM) inhibited the binding of (/sup 3/H)ADTN to both sites. Neuroleptics, however, inhibited the binding of (/sup 3/H)ADTN in two distinctly separate concentration ranges, with IC50 values of 0.15 to 40 nM at one site and 100 and 50,000 nM at the other site. The site with high affinity for dopamine and low affinity for neuroleptics had binding properties that corresponded to those of the previously characterized D3 site). The (/sup 3/H)ADTN binding site with high affinity for neuroleptics demonstrated binding characteristics similar to a site labeled by /sup 3/H-Neuroleptics. (/sup 3/H)Apomorphine appeared to label the same two sites as (/sup 3/H)ADTN, while (/sub 3/H)dopamine labeled only the D3 site. Scatchard analysis of (/sup 3/H)ADTN or (/sub 3/H)apomorphine binding, under conditions for selective labeling of the low affinity neuroleptic site (D3) and the high affinity site for neuroleptics, detected a density of 70 fmol/mg of protein for each. The density of the D3 site in the calf striatum (170 fmol/mg of protein) was much greater than that of the high affinity neuroleptic site (50 fmol/mg). In the rat, the dissociation constant (KD) of (/sup 3/H)ADTN was 2 nM for both sites. (/sup 3/H)Apomorphine, however, had a higher affinity for the D3 site (KD.1.6 nM) than for the high affinity neuroleptic site (KD.4.2 nM).

  15. PREDICTION OF ANTIGENIC AND BINDING SITES OF NEUROTOXIN 23 OF SCORPION (LYCHASMUCRONACTUS SP.)

    OpenAIRE

    Bharati K Thosare; Ingale, Arun G

    2015-01-01

    Identification of antigenic and binding site of protein is highly desirable for the design of vaccines and immunodiagnostics. The present exercise deals with a prediction of antigenic as well as binding sites of neurotoxin 23 of Lychasmucronactus. This species of scorpion having diverse molecules of toxic peptide, the peptide neurotoxin 23 is 96 amino acids long of which 23 to 96 specifically code for neurotoxin. The total of 27 such different ligand binding residue were identifie...

  16. Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay

    Institute of Scientific and Technical Information of China (English)

    SUN Xi; XU Shunqing

    2005-01-01

    A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tet rachlorodibenzo p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.

  17. Binding Mode Prediction of Evodiamine within Vanilloid Receptor TRPV1

    OpenAIRE

    Huaping Liang; Liangren Zhang; Wuzhuang Gong; Yanhui Zhang; Zhanli Wang; Lidan Sun; Hui Yu; Hongwei Jin

    2012-01-01

    Accurate assessment of the potential binding mode of drugs is crucial to computer-aided drug design paradigms. It has been reported that evodiamine acts as an agonist of the vanilloid receptor Transient receptor potential vanilloid-1 (TRPV1). However, the precise interaction between evodiamine and TRPV1 was still not fully understood. In this perspective, the homology models of TRPV1 were generated using the crystal structure of the voltage-dependent shaker family K

  18. Prediction of peptides binding to MHC class I alleles by partial periodic pattern mining

    OpenAIRE

    Meydan, Cem; Sezerman, Uğur; Sezerman, Ugur; Otu, Hasan

    2009-01-01

    MHC (Major Histocompatibility Complex) is a key player in the immune response of an organism. It is important to be able to predict which antigenic peptides will bind to a spe-cific MHC allele and which will not, creating possibilities for controlling immune response and for the applications of immunotherapy. However a problem encountered in the computational binding prediction methods for MHC class I is the presence of bulges and loops in the peptides, changing the total length. Most machine...

  19. Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: Effects on affinity for IGF-I

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.I.; D' Ercole, A.J.; Camacho-Hubner, C.; Clemmons, D.R. (Univ. of North Carolina, Chapel Hill (United States))

    1991-09-01

    The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs). The authors and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes. This observation prompted them to determine whether IGFBP-1 undergoes posttranslational modifications. Immunoprecipitation was used to show that Chinese hamster ovary cells and human hepatoma (hepG2) cells secrete {sup 32}P-labeled IGFBP-1 following incubation with ({sup 32}P)orthophosphate. Phospho amino acid analysis of {sup 32}P-labeled IGFBP-1 revealed only phosphoserine residues. A method was developed that could separate nonphosphorylated IGFBP-1 from four or five phosphorylated isoforms. These observations suggest that IGFBP-1 is secreted as a phosphoprotein and is subsequently dephosphorylated in vivo. Binding studies showed that the phosphorylated IGFBP-1 secreted by HepG2 cells has a 6-fold higher affinity for IGF-I than it does after dephosphorylation. They conclude that IGFBP-1 is phosphorylated and that this phosphorylation is a physiologically important posttranslational modification.

  20. Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins

    Science.gov (United States)

    Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

    2016-07-01

    The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10-4 M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine.The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the

  1. Binding Energy Distribution Analysis Method: Hamiltonian Replica Exchange with Torsional Flattening for Binding Mode Prediction and Binding Free Energy Estimation.

    Science.gov (United States)

    Mentes, Ahmet; Deng, Nan-Jie; Vijayan, R S K; Xia, Junchao; Gallicchio, Emilio; Levy, Ronald M

    2016-05-10

    Molecular dynamics modeling of complex biological systems is limited by finite simulation time. The simulations are often trapped close to local energy minima separated by high energy barriers. Here, we introduce Hamiltonian replica exchange (H-REMD) with torsional flattening in the Binding Energy Distribution Analysis Method (BEDAM), to reduce energy barriers along torsional degrees of freedom and accelerate sampling of intramolecular degrees of freedom relevant to protein-ligand binding. The method is tested on a standard benchmark (T4 Lysozyme/L99A/p-xylene complex) and on a library of HIV-1 integrase complexes derived from the SAMPL4 blind challenge. We applied the torsional flattening strategy to 26 of the 53 known binders to the HIV Integrase LEDGF site found to have a binding energy landscape funneled toward the crystal structure. We show that our approach samples the conformational space more efficiently than the original method without flattening when starting from a poorly docked pose with incorrect ligand dihedral angle conformations. In these unfavorable cases convergence to a binding pose within 2-3 Å from the crystallographic pose is obtained within a few nanoseconds of the Hamiltonian replica exchange simulation. We found that torsional flattening is insufficient in cases where trapping is due to factors other than torsional energy, such as the formation of incorrect intramolecular hydrogen bonds and stacking. Work is in progress to generalize the approach to handle these cases and thereby make it more widely applicable. PMID:27070865

  2. Dataset size and composition impact the reliability of performance benchmarks for peptide-MHC binding predictions

    DEFF Research Database (Denmark)

    Kim, Yohan; Sidney, John; Buus, Søren;

    2014-01-01

    cross-validation, in which all available data are iteratively split into training and testing data, and the use of blind sets generated separately from the data used to construct the predictive method. In the present study, we have compared cross-validated prediction performances generated on our last...... presence of similar peptides in the cross-validation dataset. Rather, we found that small size and low sequence/affinity diversity of either training or blind datasets were associated with large differences in cross-validated vs. blind prediction performances. We use these findings to derive quantitative...

  3. Prediction of transcription factor binding to DNA using rule induction methods

    OpenAIRE

    Huss, Mikael; Nordström, Karin

    2005-01-01

    The transcription of DNA into mRNA is initiated and aided by a number of transcription factors (TFs), proteins with DNA-binding regions that attach themselves to binding sites in the DNA (transcription factor binding sites, TFBSs). As it has become apparent that both TFs and TFBSs are highly variable, tools are needed to quantify the strength of the interaction resulting from a certain TF variant binding to a certain TFBS. We used a simple way to predict interactions between protein and DNA: ...

  4. A Novel Approach to Predict Core Residues on Cancer-Related DNA-Binding Domains

    OpenAIRE

    Ka-Chun Wong

    2016-01-01

    Protein–DNA interactions are involved in different cancer pathways. In particular, the DNA-binding domains of proteins can determine where and how gene regulatory regions are bound in different cell lines at different stages. Therefore, it is essential to develop a method to predict and locate the core residues on cancer-related DNA-binding domains. In this study, we propose a computational method to predict and locate core residues on DNA-binding domains. In particular, we have selected the ...

  5. Spectroscopic characterization of dissolved organic matter derived from different biochars and their polycylic aromatic hydrocarbons (PAHs) binding affinity.

    Science.gov (United States)

    Tang, Jianfeng; Li, Xinhu; Luo, Yan; Li, Gang; Khan, Sardar

    2016-06-01

    In recent years, biochar has received a great attention due to its high application in different sectors of environment. The feasibility of biochar applications is depended on its physical and chemical properties and biochar-derived dissolved organic matter (DOM) characteristics. This study was conducted to investigate the spectroscopic characteristics of biochar-derived DOM and its binding capacity of hydrophobic organic chemicals (HOCs). DOM solutions were isolated from five different biochars prepared through pyrolysis and analyzed for dissolved organic carbon (DOC) contents. The optical analysis with UV-visible absorption and excitation-emission matrix (EEM) fluorescence spectroscopes and DOC water distribution coefficient (KDOC) were calculated in the presence of PAHs and DOM. The DOC contents and the estimated aromaticity (SUVA254) were different for selected biochars. The DOM derived from soybean straw biochar (SBBC) showed the highest DOC contents followed by rice straw biochar (RSBC). The SBBC and RSBC peak position in the fluorescence excitation/emission matrix at longer wavelength corresponded to the peak position of other three biochars indicating that SBBC and RSBC had relatively higher degree of humification. This was well correlated with the observed KDOC values, suggesting that the KDOC value(')s dominant factor was the degree of biochar-derived DOM humification. The results of this study indicate that the optical analysis may provide valuable information regarding the characteristics of biochar-derived DOM and its application as environmental amendments for minimization of toxic organic compounds. PMID:26994600

  6. Is It Reliable to Use Common Molecular Docking Methods for Comparing the Binding Affinities of Enantiomer Pairs for Their Protein Target?

    Science.gov (United States)

    Ramírez, David; Caballero, Julio

    2016-01-01

    Molecular docking is a computational chemistry method which has become essential for the rational drug design process. In this context, it has had great impact as a successful tool for the study of ligand-receptor interaction modes, and for the exploration of large chemical datasets through virtual screening experiments. Despite their unquestionable merits, docking methods are not reliable for predicting binding energies due to the simple scoring functions they use. However, comparisons between two or three complexes using the predicted binding energies as a criterion are commonly found in the literature. In the present work we tested how wise is it to trust the docking energies when two complexes between a target protein and enantiomer pairs are compared. For this purpose, a ligand library composed by 141 enantiomeric pairs was used, including compounds with biological activities reported against seven protein targets. Docking results using the software Glide (considering extra precision (XP), standard precision (SP), and high-throughput virtual screening (HTVS) modes) and AutoDock Vina were compared with the reported biological activities using a classification scheme. Our test failed for all modes and targets, demonstrating that an accurate prediction when binding energies of enantiomers are compared using docking may be due to chance. We also compared pairs of compounds with different molecular weights and found the same results. PMID:27104528

  7. Computational prediction of heme-binding residues by exploiting residue interaction network.

    Directory of Open Access Journals (Sweden)

    Rong Liu

    Full Text Available Computational identification of heme-binding residues is beneficial for predicting and designing novel heme proteins. Here we proposed a novel method for heme-binding residue prediction by exploiting topological properties of these residues in the residue interaction networks derived from three-dimensional structures. Comprehensive analysis showed that key residues located in heme-binding regions are generally associated with the nodes with higher degree, closeness and betweenness, but lower clustering coefficient in the network. HemeNet, a support vector machine (SVM based predictor, was developed to identify heme-binding residues by combining topological features with existing sequence and structural features. The results showed that incorporation of network-based features significantly improved the prediction performance. We also compared the residue interaction networks of heme proteins before and after heme binding and found that the topological features can well characterize the heme-binding sites of apo structures as well as those of holo structures, which led to reliable performance improvement as we applied HemeNet to predicting the binding residues of proteins in the heme-free state. HemeNet web server is freely accessible at http://mleg.cse.sc.edu/hemeNet/.

  8. A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction.

    Science.gov (United States)

    Mukherjee, Somnath; Ura, Marcin; Hoey, Robert J; Kossiakoff, Anthony A

    2015-08-14

    Reversible, high-affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD~2 pM), which can be reversed by EDTA allowing controlled "capture and release" of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP-fused constructs. KD values of the Fabs were in subnanomolar to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag; thus, the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of a positive outcome. Taken together with examples of successful case specific, high-level applications like generation of conformation-, epitope- and domain-specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well-documented drawbacks. PMID:26159704

  9. Backbone and side-chain ¹H, ¹⁵N, and ¹³C resonance assignments of the microtubule-binding domain of yeast cytoplasmic dynein in the high and low-affinity states.

    Science.gov (United States)

    Takarada, Osamu; Nishida, Noritaka; Kikkawa, Masahide; Shimada, Ichio

    2014-10-01

    Cytoplasmic dynein is a motor protein that walks toward the minus end of microtubules (MTs) by utilizing the energy of ATP hydrolysis. The heavy chain of cytoplasmic dynein contains the microtubule-binding domain (MTBD). Switching of MTBD between high and low affinity states for MTs is crucial for processive movement of cytoplasmic dynein. Previous biochemical studies demonstrated that the affinity of MTBD is regulated by the AAA+ family ATPase domain, which is separated by 15 nm long coiled-coil helix. In order to elucidate the structural basis of the affinity switching mechanism of MTBD, we designed two MTBD constructs, termed MTBD-High and MTBD-Low, which are locked in high and low affinity state for MTs, respectively, by introducing a disulfide bond between the coiled-coil helix. Here, we established the backbone and side-chain assignments of MTBD-High and MTBD-Low for further structural analyses. PMID:23975349

  10. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

    Science.gov (United States)

    Larsson, Andreas; Nordlund, Paer; Jansson, Anna; Anand, Ganesh S.

    2016-01-01

    A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD). PMID:27253209

  11. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design.

    Directory of Open Access Journals (Sweden)

    Arun Chandramohan

    2016-06-01

    Full Text Available A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower. Amide hydrogen deuterium Exchange mass spectrometry (HDXMS is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD.

  12. Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    International Nuclear Information System (INIS)

    Equilibrium binding studies with the sigma receptor ligand [3H]1,3-di(2-tolyl)guanidine ([3H]DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. [Life Sci. 45:1721-1732 (1989)]. Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that [3H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [3H]DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of [3H]DTG from site 2, suggesting an association of this binding site with calcium channels

  13. Sneaking Up On The Criegee Intermediate From Below: Predicted Photoelectron Spectrum Of The CH_2OO$^-$ Anion And W3-F12 Electron Affinity Of CH$_2$OO

    CERN Document Server

    Karton, Amir; Wild, Duncan Andrew

    2013-01-01

    High level ab initio calculations were undertaken on the CH$_2$OO anion and neutral species to predict the electron affinity and anion photoelectron spectrum. The electron affinity of CH$_2$OO, \\SI{0.567}{eV}, and barrier height for dissociation of CH$_2$OO$^-$ to O$^-$ and CH$_2$O}, 16.5 kJ mol$^{-1}$, are obtained by means of the W3-F12 thermochemical protocol. Two major geometric differences between the anion and neutral, being the dihedral angle of the terminal hydrogen atoms with respect to C-O-O plane, and the O-O bond length, are reflected in the predicted spectrum as pronounced vibrational progressions.

  14. Prediction of chloride ingress and binding in cement paste

    DEFF Research Database (Denmark)

    Geiker, Mette Rica; Nielsen, Erik Pram; Herforth, Duncan

    2007-01-01

    Portland cement pastes at any content of chloride, alkalis, sulfates and carbonate was verified experimentally and found to be equally valid when applied to other data in the literature. The thermodynamic model for predicting the phase equilibria in hydrated Portland cement was introduced into an existing...... Finite Difference Model for the ingress of chlorides into concrete which takes into account its multi-component nature. The “composite theory” was then used to predict the diffusivity of each ion based on the phase assemblage present in the hydrated Portland cement paste. Agreement was found between...... steady state diffusion however. It simply implies that incremental increases in the concentration of diffusing ions in the pore solution will rapidly re-equilibrate with the hydrates present locally, where, the greater the ratio of bound to free ions, the greater the buffering effect which slows down the...

  15. SABinder: A Web Service for Predicting Streptavidin-Binding Peptides

    Science.gov (United States)

    Kang, Juanjuan; Ru, Beibei; Zhou, Peng

    2016-01-01

    Streptavidin is sometimes used as the intended target to screen phage-displayed combinatorial peptide libraries for streptavidin-binding peptides (SBPs). More often in the biopanning system, however, streptavidin is just a commonly used anchoring molecule that can efficiently capture the biotinylated target. In this case, SBPs creeping into the biopanning results are not desired binders but target-unrelated peptides (TUP). Taking them as intended binders may mislead subsequent studies. Therefore, it is important to find if a peptide is likely to be an SBP when streptavidin is either the intended target or just the anchoring molecule. In this paper, we describe an SVM-based ensemble predictor called SABinder. It is the first predictor for SBP. The model was built with the feature of optimized dipeptide composition. It was observed that 89.20% (MCC = 0.78; AUC = 0.93; permutation test, p < 0.001) of peptides were correctly classified. As a web server, SABinder is freely accessible. The tool provides a highly efficient way to exclude potential SBP when they are TUP or to facilitate identification of possibly new SBP when they are the desired binders. In either case, it will be helpful and can benefit related scientific community.

  16. SABinder: A Web Service for Predicting Streptavidin-Binding Peptides.

    Science.gov (United States)

    He, Bifang; Kang, Juanjuan; Ru, Beibei; Ding, Hui; Zhou, Peng; Huang, Jian

    2016-01-01

    Streptavidin is sometimes used as the intended target to screen phage-displayed combinatorial peptide libraries for streptavidin-binding peptides (SBPs). More often in the biopanning system, however, streptavidin is just a commonly used anchoring molecule that can efficiently capture the biotinylated target. In this case, SBPs creeping into the biopanning results are not desired binders but target-unrelated peptides (TUP). Taking them as intended binders may mislead subsequent studies. Therefore, it is important to find if a peptide is likely to be an SBP when streptavidin is either the intended target or just the anchoring molecule. In this paper, we describe an SVM-based ensemble predictor called SABinder. It is the first predictor for SBP. The model was built with the feature of optimized dipeptide composition. It was observed that 89.20% (MCC = 0.78; AUC = 0.93; permutation test, p web server, SABinder is freely accessible. The tool provides a highly efficient way to exclude potential SBP when they are TUP or to facilitate identification of possibly new SBP when they are the desired binders. In either case, it will be helpful and can benefit related scientific community. PMID:27610387

  17. Prediction of the key binding site of odorant-binding protein of Holotrichia oblita Faldermann (Coleoptera: Scarabaeida).

    Science.gov (United States)

    Zhuang, X; Wang, Q; Wang, B; Zhong, T; Cao, Y; Li, K; Yin, J

    2014-06-01

    The scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) is a predominant underground pest in the northern parts of China, and its larvae (grubs) cause great economic losses because of its wide range of host plants and covert habitats. Environmentally friendly strategies for controlling adults would have novel and broad potential applications. One potential pest management measure is the regulation of olfactory chemoreception to control target insect pests. In the process of olfactory recognition, odorant-binding proteins (OBPs) are believed to carry hydrophobic odorants from the environment to the surface of olfactory receptor neurons. To obtain a better understanding of the relationship between OBP structures and their ligands, homology modelling and molecular docking have been conducted on the interaction between HoblOBP1 and hexyl benzoate in the present study. Based on the results, site-directed mutagenesis and binding experiments were combined to describe the binding sites of HoblOBP1 and to explore its ligand-binding mechanism. After homology modelling of HoblOBP1, it was found that the three-dimensional structure of HoblOBP1 consists of six α-helices and three disulphide bridges that connect the helices, and the hydrophobic pockets are both composed of five helices. Based on the docking study, we found that van der Waals interactions and hydrophobic interactions are both important in the bonding between HoblOBP1 and hexyl benzoate. Intramolecular residues formed the hydrogen bonds in the C terminus of the protein and the bonds are crucial for the ligand-binding specificity. Finally, MET48, ILE80 and TYR111 are binding sites predicted for HoblOBP1. Using site-directed mutagenesis and fluorescence assays, it was found that ligands could not be recognized by mutant of Tyr111. A possible explanation is that the compound could not be recognized by the mutant, and remains in the binding cavity because of the loss of the intramolecular

  18. FunFOLDQA: a quality assessment tool for protein-ligand binding site residue predictions.

    Directory of Open Access Journals (Sweden)

    Daniel B Roche

    Full Text Available The estimation of prediction quality is important because without quality measures, it is difficult to determine the usefulness of a prediction. Currently, methods for ligand binding site residue predictions are assessed in the function prediction category of the biennial Critical Assessment of Techniques for Protein Structure Prediction (CASP experiment, utilizing the Matthews Correlation Coefficient (MCC and Binding-site Distance Test (BDT metrics. However, the assessment of ligand binding site predictions using such metrics requires the availability of solved structures with bound ligands. Thus, we have developed a ligand binding site quality assessment tool, FunFOLDQA, which utilizes protein feature analysis to predict ligand binding site quality prior to the experimental solution of the protein structures and their ligand interactions. The FunFOLDQA feature scores were combined using: simple linear combinations, multiple linear regression and a neural network. The neural network produced significantly better results for correlations to both the MCC and BDT scores, according to Kendall's τ, Spearman's ρ and Pearson's r correlation coefficients, when tested on both the CASP8 and CASP9 datasets. The neural network also produced the largest Area Under the Curve score (AUC when Receiver Operator Characteristic (ROC analysis was undertaken for the CASP8 dataset. Furthermore, the FunFOLDQA algorithm incorporating the neural network, is shown to add value to FunFOLD, when both methods are employed in combination. This results in a statistically significant improvement over all of the best server methods, the FunFOLD method (6.43%, and one of the top manual groups (FN293 tested on the CASP8 dataset. The FunFOLDQA method was also found to be competitive with the top server methods when tested on the CASP9 dataset. To the best of our knowledge, FunFOLDQA is the first attempt to develop a method that can be used to assess ligand binding site

  19. Phospholipase A2 and 3H-hemicholinium-3 binding sites in rat brain: A potential second-messenger role for fatty acids in the regulation of high-affinity choline uptake

    International Nuclear Information System (INIS)

    The involvement of phospholipase A2 (PLA2) and fatty acid release in the regulation of sodium-dependent high-affinity choline uptake in rat brain was assessed in vitro through the use of the specific binding of 3H-hemicholinium-3 (3H-HCh-3). Addition of arachidonic acid and other unsaturated fatty acids to rat striatal membranes in vitro resulted in a dose-dependent, temperature-independent activation of 3H-HCh-3 binding. Scatchard analysis revealed that these changes in binding result from a 2-fold increase in the affinity and capacity of 3H-HCh-3 binding. Saturated fatty acids, lysophospholipids, and phospholipids did not affect specific 3H-HCh-3 binding. Addition of defatted BSA to membranes, which had been treated previously with arachidonic acid, completely reversed the increase in specific 3H-HCh-3 binding. However, several inhibitors of fatty acid metabolism, including nordihydroguaiaretic acid, indomethacin, catalase, and superoxide dismutase, did not alter arachidonic acid-induced changes in 3H-HCh-3 binding, suggesting that unsaturated fatty acids, and not their metabolites, are directly responsible for the observed activation of specific 3H-HCh-3 binding. Additionally, unsaturated fatty acids dose-dependently inhibited high-affinity 3H-choline uptake in rat striatal synaptosomes, apparently due to the disruption of synaptosomal integrity. The phospholipase A2 inhibitors quinacrine hydrochloride, trifluoperazine, and 4-bromophenacylbromide dose-dependently inhibited potassium depolarization-induced activation of specific 3H-HCh-3 binding in slices of rat brain in vitro. Similarly, both quinacrine and trifluoperazine inhibited the metabolism of phospholipids and the release of fatty acids evoked by either elevated KCl or calcium ionophore A23187

  20. Regression applied to protein binding site prediction and comparison with classification

    Directory of Open Access Journals (Sweden)

    Gala Jean-Luc

    2009-09-01

    Full Text Available Abstract Background The structural genomics centers provide hundreds of protein structures of unknown function. Therefore, developing methods enabling the determination of a protein function automatically is imperative. The determination of a protein function can be achieved by studying the network of its physical interactions. In this context, identifying a potential binding site between proteins is of primary interest. In the literature, methods for predicting a potential binding site location generally are based on classification tools. The aim of this paper is to show that regression tools are more efficient than classification tools for patches based binding site predictors. For this purpose, we developed a patches based binding site localization method usable with either regression or classification tools. Results We compared predictive performances of regression tools with performances of machine learning classifiers. Using leave-one-out cross-validation, we showed that regression tools provide better predictions than classification ones. Among regression tools, Multilayer Perceptron ranked highest in the quality of predictions. We compared also the predictive performance of our patches based method using Multilayer Perceptron with the performance of three other methods usable through a web server. Our method performed similarly to the other methods. Conclusion Regression is more efficient than classification when applied to our binding site localization method. When it is possible, using regression instead of classification for other existing binding site predictors will probably improve results. Furthermore, the method presented in this work is flexible because the size of the predicted binding site is adjustable. This adaptability is useful when either false positive or negative rates have to be limited.

  1. Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels.

    Science.gov (United States)

    Tang, Qiong-Yao; Larry, Trevor; Hendra, Kalen; Yamamoto, Erica; Bell, Jessica; Cui, Meng; Logothetis, Diomedes E; Boland, Linda M

    2015-07-01

    All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81-104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404-404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification. PMID:25957411

  2. Binding and degradation of /sup 125/I-insulin by isolated rat renal brush border membranes: evidence for low affinity, high capacity insulin recognition sites

    Energy Technology Data Exchange (ETDEWEB)

    Meezan, E.; Pillion, D.J.; Elgavish, A.

    1988-10-01

    The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane. 125I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time- and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of 125I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane associated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of 125I-insulin by BBV, but these processes were not appreciably affected by the insulin-like growth factors IGF-I and IGF-II or by cytochrome c and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of 125I-insulin with BBV was studied at various medium osmolarities (300-1100 mosM) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of 125I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink 125I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an 125I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of 125I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10(-5) M.

  3. Binding and degradation of 125I-insulin by isolated rat renal brush border membranes: evidence for low affinity, high capacity insulin recognition sites

    International Nuclear Information System (INIS)

    The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane. 125I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time- and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of 125I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane associated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of 125I-insulin by BBV, but these processes were not appreciably affected by the insulin-like growth factors IGF-I and IGF-II or by cytochrome c and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of 125I-insulin with BBV was studied at various medium osmolarities (300-1100 mosM) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of 125I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink 125I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an 125I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of 125I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10(-5) M

  4. Age-related decrease in the responsiveness of rat articular chondrocytes to EGF is associated with diminished number and affinity for the ligand of cell surface binding sites.

    Science.gov (United States)

    Ribault, D; Habib, M; Abdel-Majid, K; Barbara, A; Mitrovic, D

    1998-01-12

    The effect of age on the responsiveness of articular chondrocytes (AC) to epidermal growth factor (EGF) was examined. Cells were isolated by digesting cartilage fragments from the humeral and femoral heads of 21-day old, 8- and 14-month old rats with collagenase. The cells were cultured under standard conditions, as monolayers. DNA synthesis was measured by [3H]thymidine incorporation and cell proliferation by the DNA content of subconfluent cultures. [125I]EGF binding and the amounts of EGF and EGF-receptor mRNAs were determined using confluent cells. DNA synthesis was decreased with age of animals. EGF stimulated DNA synthesis in cultures in 1- and 8-month old rats at low serum concentrations (< 5%), and in cultures in 14-month old animals at high serum concentrations. It also increased 5-day DNA content of cultures compared to serum-treated controls but this effect was weak in cultures in 14-month old rats. The number of high affinity binding sites for [125I]EGF decreased from 37,800 in the 1-month old to 1950 in the 14-month old rat AC. The apparent dissociation constant (Kd) also decreased with age: 0.18 nmol/l in the 1-month old; 0.12 nmol/l in the 8-month old; and 0.07 nmol/l in the 14-month old cells. AC in older rats contained more EGF mRNA and less EGF-receptor mRNA. Incubation of the cells with EGF resulted in down regulation of the EGF- and upregulation of EGF-receptor mRNA expressions. These findings show the age-related quantitative and qualitative alterations in EGF and EGF-receptor which may account, at least in part, for the diminished responsiveness of senescent AC to EGF. PMID:9509392

  5. Comparison of Different Ranking Methods in Protein-Ligand Binding Site Prediction

    Science.gov (United States)

    Gao, Jun; Liu, Qi; Kang, Hong; Cao, Zhiwei; Zhu, Ruixin

    2012-01-01

    In recent years, although many ligand-binding site prediction methods have been developed, there has still been a great demand to improve the prediction accuracy and compare different prediction algorithms to evaluate their performances. In this work, in order to improve the performance of the protein-ligand binding site prediction method presented in our former study, a comparison of different binding site ranking lists was studied. Four kinds of properties, i.e., pocket size, distance from the protein centroid, sequence conservation and the number of hydrophobic residues, have been chosen as the corresponding ranking criterion respectively. Our studies show that the sequence conservation information helps to rank the real pockets with the most successful accuracy compared to others. At the same time, the pocket size and the distance of binding site from the protein centroid are also found to be helpful. In addition, a multi-view ranking aggregation method, which combines the information among those four properties, was further applied in our study. The results show that a better performance can be achieved by the aggregation of the complementary properties in the prediction of ligand-binding sites. PMID:22942732

  6. Rearrangements of α-helical structures of FlgN chaperone control the binding affinity for its cognate substrates during flagellar type III export.

    Science.gov (United States)

    Kinoshita, Miki; Nakanishi, Yuki; Furukawa, Yukio; Namba, Keiichi; Imada, Katsumi; Minamino, Tohru

    2016-08-01

    The bacterial flagellar type III export chaperones not only act as bodyguards to protect their cognate substrates from aggregation and proteolysis in the cytoplasm but also ensure the order of export through their interactions with an export gate protein FlhA. FlgN chaperone binds to FlgK and FlgL with nanomolar affinity and transfers them to FlhA for their efficient and rapid transport for the formation of the hook-filament junction zone. However, it remains unknown how FlgN releases FlgK and FlgL at the FlhA export gate platform in a timely manner. Here, we have solved the crystal structure of Salmonella FlgN at 2.3 Å resolution and carried out structure-based functional analyses. FlgN consists of three α helices, α1, α2 and α3. Helix α1 adopts two distinct, extended and bent conformations through the conformational change of N-loop between α1 and α2. The N-loop deletion not only increases the probability of FlgN dimer formation but also abolish the interaction between FlgN and FlgK. Highly conserved Asn-92, Asn-95 and Ile-103 residues in helix α3 are involved in the strong interaction with FlgK. We propose that the N-loop coordinates helical rearrangements of FlgN with the association and dissociation of its cognate substrates during their export. PMID:27178222

  7. The Synthesis of 1,4,7-Triazacyclononane Conjugated Amyloid-phillic Compound and Its Binding Affinity to the β-Amyloid Fibril

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Jong Min; Jo, Jee Hye [Sejong University, Seoul (Korea, Republic of)

    2003-09-15

    The development of new compounds which have affinity for the β-amyloid fibril would lead to the new compounds that could have therapeutic effects on AD. Previously, we generated new amyloid-phillic amide derivative of Chrysamine G and found that this compound protect human astrocyte cells against Aβ-induced toxicity. As conjugation of amyloid-philic molecules with suitable metal chelating ligands could lead to new diagnostic molecules for in vivo quantification of amyloid deposition and new probes for amyloid structure, we designed the compound, which was conjugate of 1,4,7-triazacyclo-nonane and the amyloid-philic compound 1. Here, we would like to report the synthesis of compound 2 and its binding property of β-amyloid fibril. The synthesis of compound was achieved by combining three fragments the biphenyl amine, isophthalic acid 5 and 1,4,7-triazacyclononane. The synthesis of isophthalic acid part 5 commenced with esterfication of isophthalic acid in methanol with HCl to produce the monoester 4 in 38% yield. Treatment of the monoester 4 with oxalyl chloride afforded the acyl chloride 5 in 95% yield.

  8. The N-Terminal T-T Motif of a Third-Generation HIV-1 Fusion Inhibitor Is Not Required for Binding Affinity and Antiviral Activity.

    Science.gov (United States)

    Chong, Huihui; Qiu, Zonglin; Su, Yang; He, Yuxian

    2015-08-27

    The highlighted next-generation HIV-1 fusion inhibitor peptide 1 is capped by two threonines. Here, we generated peptide 2 by deleting the T-T motif and compared their structural and antiviral properties. Significantly, two peptides showed similar helical and oligomeric states in solution, comparable binding affinities to the target, and no significant difference to inhibit HIV-1 fusion and infection. Also, the T-T motif was not associated with peptide 1 resistant mutations and its deletion did not affect peptide 1 against enfuvirtide-resistant HIV-1 mutants. The redundancy of the T-T motif was further verified by the model peptide C34 and short peptide inhibitors that mainly target the gp41 pocket, suggesting that the N-terminal T-T motif of peptide 1 could be removed or modified toward the development of new anti-HIV-1 drugs. Consistently, our data have verified that the M-T hook structure rather than the T-T motif is an efficient strategy for short peptide fusion inhibitors. PMID:26256053

  9. Acceleration of wound healing in acute full-thickness skin wounds using a collagen-binding peptide with an affinity for MSCs

    Directory of Open Access Journals (Sweden)

    Huili Wang

    2014-10-01

    Full Text Available Mesenchymal stem cells (MSCs have been accepted as a promising cell source in tissue repair and regeneration. However, the inability to enrich MSCs in target areas limits their wide application. As a result, it has been a major goal to induce MSCs to be abundantly and specifically recruited to the injury site. In this study, a peptide with a specific affinity for MSCs (E7 peptide was immobilized to a collagen scaffold via a collagen-binding domain (CBD to construct a functional collagen scaffold. In addition, the hypothesis that this method could recruit MSCs specifically was evaluated in a porcine model. In vivo investigations indicated that due to the immunoreaction, the CBD-MSC-peptide collagen scaffold enhanced MSC adhesion and infiltration and promoted wound healing. At day 7 after surgery, we found more infiltrating cells and capillaries in the Collagen/CBD-E7 peptide group compared to the Scaffold group. At day 14, 21 and 28, a faster healing process was observed in the Collagen/CBD-E7 peptide group, with significant differences compared with the other groups (P < 0.05, P < 0.01. The results demonstrate the potential use of targeted therapy to rapidly heal skin wounds.

  10. NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Blicher, Thomas;

    2007-01-01

    surpassed 1500. Characterizing the specificity of each separately would be a major undertaking. PRINCIPAL FINDINGS: Here, we have drawn on a large database of known peptide-HLA-I interactions to develop a bioinformatics method, which takes both peptide and HLA sequence information into account, and...... generates quantitative predictions of the affinity of any peptide-HLA-I interaction. Prospective experimental validation of peptides predicted to bind to previously untested HLA-I molecules, cross-validation, and retrospective prediction of known HIV immune epitopes and endogenous presented peptides, all...... provide new basic insights into HLA structure-function relationships. The method is available at http://www.cbs.dtu.dk/services/NetMHCpan....

  11. Accuracy of binding mode prediction with a cascadic stochastic tunneling method.

    Science.gov (United States)

    Fischer, Bernhard; Basili, Serena; Merlitz, Holger; Wenzel, Wolfgang

    2007-07-01

    We investigate the accuracy of the binding modes predicted for 83 complexes of the high-resolution subset of the ASTEX/CCDC receptor-ligand database using the atomistic FlexScreen approach with a simple forcefield-based scoring function. The median RMS deviation between experimental and predicted binding mode was just 0.83 A. Over 80% of the ligands dock within 2 A of the experimental binding mode, for 60 complexes the docking protocol locates the correct binding mode in all of ten independent simulations. Most docking failures arise because (a) the experimental structure clashed in our forcefield and is thus unattainable in the docking process or (b) because the ligand is stabilized by crystal water. PMID:17427957

  12. Prediction of heme binding residues from protein sequences with integrative sequence profiles

    OpenAIRE

    2012-01-01

    Background The heme-protein interactions are essential for various biological processes such as electron transfer, catalysis, signal transduction and the control of gene expression. The knowledge of heme binding residues can provide crucial clues to understand these activities and aid in functional annotation, however, insufficient work has been done on the research of heme binding residues from protein sequence information. Methods We propose a sequence-based approach for accurate prediction...

  13. G-protein-coupled receptor affinity prediction based on the use of a profiling dataset: QSAR design, synthesis, and experimental validation.

    Science.gov (United States)

    Rolland, Catherine; Gozalbes, Rafael; Nicolaï, Eric; Paugam, Marie-France; Coussy, Laurent; Barbosa, Frédérique; Horvath, Dragos; Revah, Frédéric

    2005-10-20

    A QSAR model accounting for "average" G-protein-coupled receptor (GPCR) binding was built from a large set of experimental standardized binding data (1939 compounds systematically tested over 40 different GPCRs) and applied to the design of a library of "GPCR-predicted" compounds. Three hundred and sixty of these compounds were randomly selected and tested in 21 GPCR binding assays. Positives were defined by their ability to inhibit by more than 70% the binding of reference compounds at 10 microM. A 5.5-fold enrichment in positives was observed when comparing the "GPCR-predicted" compounds with 600 randomly selected compounds predicted as "non-GPCR" from a general collection. The model was efficient in predicting strongest binders, since enrichment was greater for higher cutoffs. Significant enrichment was also observed for peptidic GPCRs and receptors not included to develop the QSAR model, suggesting the usefulness of the model to design ligands binding with newly identified GPCRs, including orphan ones. PMID:16220973

  14. Characterization of the binding of a novel nonxanthine adenosine antagonist radioligand, ( sup 3 H)CGS 15943, to multiple affinity states of the adenosine A1 receptor in the rat cortex

    Energy Technology Data Exchange (ETDEWEB)

    Jarvis, M.F.; Williams, M.; Do, U.H.; Sills, M.A. (CIBA-GEIGY Corp., Summit, NJ (USA))

    1991-01-01

    The triazoloquinazoline CGS 15943 is the first reported nonxanthine adenosine antagonist that has high affinity for brain adenosine receptors. In the present study, the binding of (3H) CGS 15943 to recognition sites in rat cortical membranes was characterized. Saturation experiments revealed that (3H)CGS 15943 labeled a single class of recognition sites with high affinity and limited capacity. Competition studies revealed that the binding of (3H)CGS 15943 was consistent with the labeling of brain adenosine A1 receptors. Adenosine agonists inhibited 1 nM (3H)CGS 15943 binding with the following order of activity N6-cyclopentyladenosine (IC50 = 15 nM) greater than 2-chloroadenosine greater than (R)-N6-phenylisopropyladenosine greater than 5'-N6-ethylcarboxamidoadenosine greater than (S)N6-phenylisopropyladenosine greater than CGS 21680 greater than CV 1808 (IC50 greater than 10,000 nM). The potency order for adenosine antagonists was CGS 15943 (IC50 = 5 nM) greater than 8-phenyltheophylline greater than 1,3-dipropyl-8-(4-amino-2-chloro)phenylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than theophylline = caffeine (IC50 greater than 10,000 nM). Antagonist inhibition curves were steep and best described by a one-site binding model. In contrast, adenosine A1 agonist competition curves were shallow, as indicated by Hill coefficients less than unity. Computer analysis revealed that these inhibition curves were best described by a two-site binding model. Agonist competition curves generated in the presence of 1 mM GTP resulted in a rightward shift and steepening of the inhibition-concentration curves, whereas antagonist binding was not altered in the presence of GTP. The complex binding interactions found with adenosine agonists indicate that (3H)CGS 15943 labels both high and low affinity components of the adenosine A1 receptor in the rat cortex.

  15. Calciomics:prediction and analysis of EF-hand calcium binding proteins by protein engineering

    Institute of Scientific and Technical Information of China (English)

    YANG; Jenny; Jie

    2010-01-01

    Ca2+ plays a pivotal role in the physiology and biochemistry of prokaryotic and mammalian organisms.Viruses also utilize the universal Ca2+ signal to create a specific cellular environment to achieve coexistence with the host,and to propagate.In this paper we first describe our development of a grafting approach to understand site-specific Ca2+ binding properties of EF-hand proteins with a helix-loop-helix Ca2+ binding motif,then summarize our prediction and identification of EF-hand Ca2+ binding sites on a genome-wide scale in bacteria and virus,and next report the application of the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor(Shr) of Streptococcus pyrogenes and the nonstructural protein 1(nsP1) of Sindbis virus.When methods such as the grafting approach are developed in conjunction with prediction algorithms we are better able to probe continuous Ca2+-binding sites that have been previously underrepresented due to the limitation of conventional methodology.

  16. Thyroid hormones and glucocorticoids act synergistically in the regulation of the low affinity glucocorticoid binding sites in the male rat liver.

    Science.gov (United States)

    Chirino, R; Fernández, L; López, A; Navarro, D; Rivero, J F; Díaz-Chico, J C; Díaz-Chico, B N

    1991-12-01

    The low affinity glucocorticoid binding sites (LAGS) have been described and partially characterized in both the nuclei and microsomes of rat liver. The LAGS concentration is under endocrine regulation, as proved by their decrease after adrenalectomy and their almost complete disappearance after hypophysectomy. This article describes new data that also implicate the thyroid hormones in the endocrine regulation of LAGS. The LAGS were measured by [3H]dexamethasone exchange assay in crude microsome suspensions of rat liver. Propylthiouracil-induced hypothyroidism (TX) provoked a 90% reduction in the LAGS levels with respect to the control value. The administration of T3 to TX rats was able to completely restore the LAGS level. On the other hand, adrenalectomy (ADX) provoked a 50% decrease in LAGS levels, and this effect could be reverted by treatment with corticosterone acetate. TX rats that were also adrenalectomized (TX-ADX) showed a LAGS level similar to that of the TX rats. However, treatment of these rats with T3 was much less effective than in TX rats. A complete restoration of the LAGS level in TX-ADX rats could be achieved only with a combined treatment of corticosterone acetate plus T3. Similar results to those obtained in TX-ADX rats were also obtained in immature or hypophysectomized rats, two experimental models known to possess very low or undetectable levels of LAGS. From these findings we conclude that: 1) thyroid hormones, as well as glucocorticoids, play an important role in the regulation of the LAGS level; 2) glucocorticoids and thyroid hormones act synergistically in the endocrine regulation of LAGS; and 3) the results obtained in the hypophysectomized rats point to a direct action of glucocorticoids and T3 on the LAGS level of the rat liver. PMID:1954893

  17. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone.

    Science.gov (United States)

    Shabanpoor, Fazel; Bathgate, Ross A D; Belgi, Alessia; Chan, Linda J; Nair, Vinojini B; Wade, John D; Hossain, Mohammed Akhter

    2012-04-01

    Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide. PMID:22425984

  18. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

    International Nuclear Information System (INIS)

    Highlights: ► A mono-Eu-DTPA conjugated peptide ligand, Eu-DTPA-(A)-H2, has been developed. ► The choice of a site for incorporation of a chelator is critical. ► The labeled peptide retains full activity at the RXFP1 receptor. ► It is markedly cheaper to produce and easier to use than radioactive probes. -- Abstract: Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure–activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand–receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of

  19. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

    Energy Technology Data Exchange (ETDEWEB)

    Shabanpoor, Fazel [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia); Bathgate, Ross A.D.; Belgi, Alessia [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC 3010 (Australia); Chan, Linda J.; Nair, Vinojini B.; Wade, John D. [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia); Hossain, Mohammed Akhter, E-mail: akhter.hossain@florey.edu.au [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer A mono-Eu-DTPA conjugated peptide ligand, Eu-DTPA-(A)-H2, has been developed. Black-Right-Pointing-Pointer The choice of a site for incorporation of a chelator is critical. Black-Right-Pointing-Pointer The labeled peptide retains full activity at the RXFP1 receptor. Black-Right-Pointing-Pointer It is markedly cheaper to produce and easier to use than radioactive probes. -- Abstract: Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to

  20. PREDICTION OF ANTIGENIC AND BINDING SITES OF NEUROTOXIN 23 OF SCORPION (LYCHASMUCRONACTUS SP.

    Directory of Open Access Journals (Sweden)

    Bharati K Thosare

    2015-07-01

    Full Text Available Identification of antigenic and binding site of protein is highly desirable for the design of vaccines and immunodiagnostics. The present exercise deals with a prediction of antigenic as well as binding sites of neurotoxin 23 of Lychasmucronactus. This species of scorpion having diverse molecules of toxic peptide, the peptide neurotoxin 23 is 96 amino acids long of which 23 to 96 specifically code for neurotoxin. The total of 27 such different ligand binding residue were identified by ConSurf and Raptor X server. The web tool Ellipro which implements Modeller and Jmol viewer, predicted and visualized the linear and discontinuous antibody epitopes ofneurotoxin 23 protein sequence.Thus the information discussed here provides a clue for understanding antigenic site and molecular function of neurotoxin 23.

  1. How well do lipophilicity parameters, MEEKC microemulsion capacity factor, and plasma protein binding predict CNS tissue binding?

    Science.gov (United States)

    Zamek-Gliszczynski, Maciej J; Sprague, Karen E; Espada, Alfonso; Raub, Thomas J; Morton, Stuart M; Manro, Jason R; Molina-Martin, Manuel

    2012-05-01

    Brain fraction unbound (Fu) is critical to understanding the pharmacokinetics/dynamics of central nervous system (CNS) drugs, thus several surrogate predictors have been proposed. At present, correlations between brain Fu, microemulsion electrokinetic chromatography capacity factor (MEEKC k'), plasma Fu, octanol-water partition coefficient (clogP), and LogP at pH 7.4 (clogD(7.4) ) were compared for 94 diverse molecules, and additionally for 587 compounds. MEEKC k' was a better predictor of brain Fu (r(2) = 0.74) than calculated lipophilicity parameters (clogP r(2) = 0.51-0.54, clogD(7.4) r(2) = 0.41-0.44), but it was not superior to plasma Fu (r(2) = 0.74-0.85) as a predictor of brain Fu. MEEKC k' did not predict plasma Fu(r(2) = 0.58) as well as brain Fu, and the extent of improvement over clogP or clogD(7.4) (r(2) = 0.41-0.49) was less pronounced. Although log-log-correlation analysis supported seemingly strong prediction of brain Fu both by MEEKC k' and by plasma Fu (r(2) ≥ 0.74), analysis of prediction error estimated a 10-fold and 6.9-8.6-fold prediction interval for brain Fu estimated using MEEKC k' and plasma Fu, respectively. Therefore, MEEKC k' and plasma Fu can predict the log order of CNS tissue binding, but they cannot provide truly quantitative brain Fu predictions necessary to support in-vitro-to-in-vivo extrapolations and pharmacokinetic/dynamic data interpretation. PMID:22344827

  2. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    Energy Technology Data Exchange (ETDEWEB)

    Orcutt, Kelly Davis; Slusarczyk, Adrian L. [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Cieslewicz, Maryelise [Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Ruiz-Yi, Benjamin [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Bhushan, Kumar R. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Frangioni, John V. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Wittrup, K. Dane, E-mail: wittrup@mit.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2011-02-15

    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2{+-}1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a {sup 111}In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  3. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    International Nuclear Information System (INIS)

    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2±1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  4. Affinity Purification of Insulin by Peptide-Ligand Affinity Chromatography

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The affinity heptapeptide (HWWWPAS) for insulin, selected from phage display library,was coupled to EAH Sepharose 4B gel and packed to a 1-mL column. The column was used for the affinity purification of insulin from protein mixture and commercial insulin preparation. It was observed that the minor impurity in the commercial insulin was removed by the affinity chromatography. Nearly 40 mg of insulin could be purified with the 1-mL affinity column. The results revealed the high specificity and capacity of the affinity column for insulin purification. Moreover, based on the analysis of the amino acids in the peptide sequence, shorter peptides were designed and synthesized for insulin chromatography. As a result, HWWPS was found to be a good alternative to HWWWPAS, while the other two peptides with three or four amino acids showed weak affinity for insulin. The results indicated that the peptide sequence of HWWWPAS was quite conservative for specific binding of insulin.

  5. Improving the prediction of protein binding sites by combining heterogeneous data and Voronoi diagrams

    Directory of Open Access Journals (Sweden)

    Fernandez-Fuentes Narcis

    2011-08-01

    Full Text Available Abstract Background Protein binding site prediction by computational means can yield valuable information that complements and guides experimental approaches to determine the structure of protein complexes. Predictions become even more relevant and timely given the current resolution of protein interaction maps, where there is a very large and still expanding gap between the available information on: (i which proteins interact and (ii how proteins interact. Proteins interact through exposed residues that present differential physicochemical properties, and these can be exploited to identify protein interfaces. Results Here we present VORFFIP, a novel method for protein binding site prediction. The method makes use of broad set of heterogeneous data and defined of residue environment, by means of Voronoi Diagrams that are integrated by a two-steps Random Forest ensemble classifier. Four sets of residue features (structural, energy terms, sequence conservation, and crystallographic B-factors used in different combinations together with three definitions of residue environment (Voronoi Diagrams, sequence sliding window, and Euclidian distance have been analyzed in order to maximize the performance of the method. Conclusions The integration of different forms information such as structural features, energy term, evolutionary conservation and crystallographic B-factors, improves the performance of binding site prediction. Including the information of neighbouring residues also improves the prediction of protein interfaces. Among the different approaches that can be used to define the environment of exposed residues, Voronoi Diagrams provide the most accurate description. Finally, VORFFIP compares favourably to other methods reported in the recent literature.

  6. Proteins and Their Interacting Partners: An Introduction to Protein–Ligand Binding Site Prediction Methods

    Directory of Open Access Journals (Sweden)

    Daniel Barry Roche

    2015-12-01

    Full Text Available Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein–ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein–ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein–ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP and the Continuous Automated Model EvaluatiOn (CAMEO projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

  7. The peripheral benzodiazepine receptor ligand PK11195 binds with high affinity to the acute phase reactant α1-acid glycoprotein: implications for the use of the ligand as a CNS inflammatory marker

    International Nuclear Information System (INIS)

    The peripheral benzodiazepine receptor ligand PK11195 has been used as an in vivo marker of neuroinflammation in positron emission tomography studies in man. One of the methodological issues surrounding the use of the ligand in these studies is the highly variable kinetic behavior of [11C]PK11195 in plasma. We therefore undertook a study to measure the binding of [3H]PK11195 to whole human blood and found a low level of binding to blood cells but extensive binding to plasma proteins. Binding assays using [3H]PK11195 and purified human plasma proteins demonstrated a strong binding to α1-acid glycoprotein (AGP) and a much weaker interaction with albumin. Immunodepletion of AGP from plasma resulted in the loss of plasma [3H]PK11195 binding demonstrating: (i) the specificity of the interaction and (ii) that AGP is the major plasma protein to which PK11195 binds with high affinity. PK11195 was able to displace fluorescein-dexamethasone from AGP with IC50 of 11C]PK11195 to the brain parenchyma in diseases with blood brain barrier breakdown. Finally, local synthesis of AGP at the site of brain injury may contribute the pattern of [11C]PK11195 binding observed in neuroinflammatory diseases

  8. Genome-wide de novo prediction of cis-regulatory binding sites in prokaryotes

    Science.gov (United States)

    Zhang, Shaoqiang; Xu, Minli; Su, Zhengchang

    2009-01-01

    Although cis-regulatory binding sites (CRBSs) are at least as important as the coding sequences in a genome, our general understanding of them in most sequenced genomes is very limited due to the lack of efficient and accurate experimental and computational methods for their characterization, which has largely hindered our understanding of many important biological processes. In this article, we describe a novel algorithm for genome-wide de novo prediction of CRBSs with high accuracy. We designed our algorithm to circumvent three identified difficulties for CRBS prediction using comparative genomics principles based on a new method for the selection of reference genomes, a new metric for measuring the similarity of CRBSs, and a new graph clustering procedure. When operon structures are correctly predicted, our algorithm can predict 81% of known individual binding sites belonging to 94% of known cis-regulatory motifs in the Escherichia coli K12 genome, while achieving high prediction specificity. Our algorithm has also achieved similar prediction accuracy in the Bacillus subtilis genome, suggesting that it is very robust, and thus can be applied to any other sequenced prokaryotic genome. When compared with the prior state-of-the-art algorithms, our algorithm outperforms them in both prediction sensitivity and specificity. PMID:19383880

  9. Sequence-based prediction of protein-peptide binding sites using support vector machine.

    Science.gov (United States)

    Taherzadeh, Ghazaleh; Yang, Yuedong; Zhang, Tuo; Liew, Alan Wee-Chung; Zhou, Yaoqi

    2016-05-15

    Protein-peptide interactions are essential for all cellular processes including DNA repair, replication, gene-expression, and metabolism. As most protein-peptide interactions are uncharacterized, it is cost effective to investigate them computationally as the first step. All existing approaches for predicting protein-peptide binding sites, however, are based on protein structures despite the fact that the structures for most proteins are not yet solved. This article proposes the first machine-learning method called SPRINT to make Sequence-based prediction of Protein-peptide Residue-level Interactions. SPRINT yields a robust and consistent performance for 10-fold cross validations and independent test. The most important feature is evolution-generated sequence profiles. For the test set (1056 binding and non-binding residues), it yields a Matthews' Correlation Coefficient of 0.326 with a sensitivity of 64% and a specificity of 68%. This sequence-based technique shows comparable or more accurate than structure-based methods for peptide-binding site prediction. SPRINT is available as an online server at: http://sparks-lab.org/. © 2016 Wiley Periodicals, Inc. PMID:26833816

  10. Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

    Science.gov (United States)

    Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

    2014-01-01

    ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an

  11. A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules

    DEFF Research Database (Denmark)

    Pandya, Mital; Rasmussen, Michael; Hansen, Andreas;

    2015-01-01

    pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network......-based predictions (NetMHCpan). The updated NetMHCpan server was used to predict BoLA-I binding peptides within the P1 structural polyprotein sequence of FMDV (strain A24 Cruzeiro) for BoLA-1*01901, BoLA-2*00801, BoLA-2*01201, and BoLA-4*02401. Peptide binding affinity and stability were determined for these Bo....... The results of these analyses showed that BoLA alleles cluster into three distinct groups with the potential to define “BoLA supertypes.” This streamlined approach identifies potential T cell epitopes from pathogens, such as FMDV, and provides insight into T cell immunity following infection or vaccination....

  12. MULTIPRED2: A computational system for large-scale identification of peptides predicted to bind to HLA supertypes and alleles

    DEFF Research Database (Denmark)

    Zhang, Guang Lan; DeLuca, David S.; Keskin, Derin B.;

    2011-01-01

    MULTIPRED2 is a computational system for facile prediction of peptide binding to multiple alleles belonging to human leukocyte antigen (HLA) class I and class II DR molecules. It enables prediction of peptide binding to products of individual HLA alleles, combination of alleles, or HLA supertypes...

  13. Machine learning competition in immunology – Prediction of HLA class I binding peptides

    DEFF Research Database (Denmark)

    Zhang, Guang Lan; Ansari, Hifzur Rahman; Bradley, Phil;

    2011-01-01

    ., 2008] and [Larsen et al., 2010]). HTMS involves HLA typing, immunoaffinity chromatography of HLA molecules, HLA extraction, and chromatography combined with tandem mass spectrometry, followed by the application of computational algorithms for peptide characterization (Bassani-Sternberg et al., 2010......). Hundreds of naturally processed HLA class I associated peptides have been identified in individual studies using HTMS in normal (Escobar et al., 2008), cancer ( [Antwi et al., 2009] and [Bassani-Sternberg et al., 2010]), autoimmunity-related (Ben Dror et al., 2010), and infected samples (Wahl et al, 2010...... of peptide binding, therefore, determines the accuracy of the overall method. Computational predictions of peptide binding to HLA, both class I and class II, use a variety of algorithms ranging from binding motifs to advanced machine learning techniques ( [Brusic et al., 2004] and [Lafuente and Reche...

  14. Computational prediction of cAMP receptor protein (CRP) binding sites in cyanobacterial genomes

    Science.gov (United States)

    Xu, Minli; Su, Zhengchang

    2009-01-01

    Background Cyclic AMP receptor protein (CRP), also known as catabolite gene activator protein (CAP), is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The loss of CRPs in these species

  15. Computational prediction of cAMP receptor protein (CRP binding sites in cyanobacterial genomes

    Directory of Open Access Journals (Sweden)

    Su Zhengchang

    2009-01-01

    Full Text Available Abstract Background Cyclic AMP receptor protein (CRP, also known as catabolite gene activator protein (CAP, is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The

  16. Genome-wide de novo prediction of cis-regulatory binding sites in prokaryotes

    OpenAIRE

    Zhang, Shaoqiang; Xu, Minli; Li, Shan; Su, Zhengchang

    2009-01-01

    Although cis-regulatory binding sites (CRBSs) are at least as important as the coding sequences in a genome, our general understanding of them in most sequenced genomes is very limited due to the lack of efficient and accurate experimental and computational methods for their characterization, which has largely hindered our understanding of many important biological processes. In this article, we describe a novel algorithm for genome-wide de novo prediction of CRBSs with high accuracy. We desi...

  17. Further characterization of a high affinity thyrotropin binding site on the rat thyrotropin receptor which is an epitope for blocking antibodies from idiopathic myxedema patients but not thyroid stimulating antibodies from Graves' patients.

    Science.gov (United States)

    Kosugi, S; Ban, T; Akamizu, T; Kohn, L D

    1991-10-31

    Cysteine 390 of the rat thyrotropin (TSH) receptor, when mutated to serine, results in a receptor with a reduced ability of TSH to bind and increase cAMP levels but a preserved ability of thyroid stimulating autoantibodies (TSAbs) from hyperthyroid Graves' patients to increase cAMP levels. The ability of receptor autoantibodies from hypothyroid patients with idiopathic myxedema to inhibit the TSAb activity which is preserved is, however, like TSH binding, significantly reduced. Cysteine 390, together with tyrosine 385, thus appears to be an important determinant in a high affinity TSH binding site which is an epitope for receptor autoantibodies which block TSH or TSAb action and cause hypothyroidism rather than TSAbs which increase cAMP levels and are associated with hyperthyroidism. Threonine 388 and aspartic acid 403 may contribute to this ligand interaction site. PMID:1719963

  18. Quantitative prediction of peptide binding to HLA-DP1 protein.

    Science.gov (United States)

    Ivanov, Stefan; Dimitrov, Ivan; Doytchinova, Irini

    2013-01-01

    The exogenous proteins are processed by the host antigen-processing cells. Peptidic fragments of them are presented on the cell surface bound to the major hystocompatibility complex (MHC) molecules class II and recognized by the CD4+ T lymphocytes. The MHC binding is considered as the crucial prerequisite for T-cell recognition. Only peptides able to form stable complexes with the MHC proteins are recognized by the T-cells. These peptides are known as T-cell epitopes. All T-cell epitopes are MHC binders, but not all MHC binders are T-cell epitopes. The T-cell epitope prediction is one of the main priorities of immunoinformatics. In the present study, three chemometric techniques are combined to derive a model for in silico prediction of peptide binding to the human MHC class II protein HLA-DP1. The structures of a set of known peptide binders are described by amino acid z-descriptors. Data are processed by an iterative self-consisted algorithm using the method of partial least squares, and a quantitative matrix (QM) for peptide binding prediction to HLA-DP1 is derived. The QM is validated by two sets of proteins and showed an average accuracy of 86 percent. PMID:24091413

  19. Predicting the binding patterns of hub proteins: a study using yeast protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Carson M Andorf

    Full Text Available BACKGROUND: Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH with one or two binding sites, or multiple-interface hubs (MIH with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations or party hubs (i.e., simultaneously interact with multiple partners. METHODOLOGY: Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques. CONCLUSIONS: Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions. AVAILABILITY: We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.

  20. A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules

    Science.gov (United States)

    Major histocompatibility complex (MHC) class I molecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8positive T-cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presenta...

  1. A Theoretical Study of the Relationships between Electronic Structure and CB1 and CB2 Cannabinoid Receptor Binding Affinity in a Group of 1-Aryl-5-(1-H-pyrrol-1-yl-1-H-pyrazole-3-carboxamides

    Directory of Open Access Journals (Sweden)

    Francisco Salgado-Valdés

    2014-01-01

    Full Text Available We report the results of a search for model-based relationships between hCB1 and hCB2 receptor binding affinity and molecular structure for a group of 1-aryl-5-(1-H-pyrrol-1-yl-1-H-pyrazole-3-carboxamides. The wave functions and local atomic reactivity indices were obtained at the B3LYP/6-31G(d,p levels of theory with full geometry optimization. Interaction pharmacophores were generated for both receptors. The main conclusions of this work are as follows. (1 We obtained statistically significant equations relating the variation of hCB1 and hCB2 receptor binding affinities with the variation of definite sets of local atomic reactivity indices. (2 The interaction of the molecules with the hCB1 and hCB2 receptors seems to be highly complex and mainly orbital controlled. (3 The interaction mechanisms seem to be different for each type of receptor. This study, contrarily to the statistically backed ones, is able to provide a microscopic insight of the mechanisms involved in the binding process.

  2. Mono-anionic Phosphopeptides Produced by Unexpected Histidine Alkylation Exhibit High Plk1 Polo-box Domain-binding Affinities and Enhanced Antiproliferative Effects in HeLa Cells

    Science.gov (United States)

    Qian, Wen-Jian; Park, Jung-Eun; Lim, Dan; Lai, Christopher C.; Kelley, James A.; Park, Suk-Youl; Lee, Ki-Won; Yaffe, Michael B.; Lee, Kyung S.; Burke, Terrence R.

    2016-01-01

    Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current paper we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics. PMID:25283071

  3. Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.

    Science.gov (United States)

    Qian, Wen-Jian; Park, Jung-Eun; Lim, Dan; Lai, Christopher C; Kelley, James A; Park, Suk-Youl; Lee, Ki Won; Yaffe, Michael B; Lee, Kyung S; Burke, Terrence R

    2014-11-01

    Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics. PMID:25283071

  4. Predicting DNA-binding sites of proteins from amino acid sequence

    Directory of Open Access Journals (Sweden)

    Wu Feihong

    2006-05-01

    Full Text Available Abstract Background Understanding the molecular details of protein-DNA interactions is critical for deciphering the mechanisms of gene regulation. We present a machine learning approach for the identification of amino acid residues involved in protein-DNA interactions. Results We start with a Naïve Bayes classifier trained to predict whether a given amino acid residue is a DNA-binding residue based on its identity and the identities of its sequence neighbors. The input to the classifier consists of the identities of the target residue and 4 sequence neighbors on each side of the target residue. The classifier is trained and evaluated (using leave-one-out cross-validation on a non-redundant set of 171 proteins. Our results indicate the feasibility of identifying interface residues based on local sequence information. The classifier achieves 71% overall accuracy with a correlation coefficient of 0.24, 35% specificity and 53% sensitivity in identifying interface residues as evaluated by leave-one-out cross-validation. We show that the performance of the classifier is improved by using sequence entropy of the target residue (the entropy of the corresponding column in multiple alignment obtained by aligning the target sequence with its sequence homologs as additional input. The classifier achieves 78% overall accuracy with a correlation coefficient of 0.28, 44% specificity and 41% sensitivity in identifying interface residues. Examination of the predictions in the context of 3-dimensional structures of proteins demonstrates the effectiveness of this method in identifying DNA-binding sites from sequence information. In 33% (56 out of 171 of the proteins, the classifier identifies the interaction sites by correctly recognizing at least half of the interface residues. In 87% (149 out of 171 of the proteins, the classifier correctly identifies at least 20% of the interface residues. This suggests the possibility of using such classifiers to identify

  5. High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4(+) T cell responses more than 30 years post-vaccinia virus vaccination

    DEFF Research Database (Denmark)

    Wang, M.; Tang, Sheila Tuyet; Lund, Ole; Dziegiel, M. H.; Buus, S.; Claesson, M. H.

    2009-01-01

    Interferon-gamma secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human leucoc...

  6. The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells

    DEFF Research Database (Denmark)

    Honoré, B; Vorum, H

    2000-01-01

    quite diverse invertebrate organisms such as DCB-45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB-39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca(2+) affinity is rather low with dissociation constants around 10(-4)-10(-3) M. The proteins may participate in Ca...

  7. Synthesis of a Hoechst 32258 analogue amino acid building block for direct incorporation of a fluorescent, high-affinity DNA binding motif into peptides

    DEFF Research Database (Denmark)

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