Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor.

PubMed

Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Gorska, Magdalena; Tuszynski, Jack A; Wozniak, Michal

2016-01-19

Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding.

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

PubMed

Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

2016-01-01

Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

Face-name learning in older adults: a benefit of hyper-binding.

PubMed

Weeks, Jennifer C; Biss, Renée K; Murphy, Kelly J; Hasher, Lynn

2016-10-01

Difficulty remembering faces and corresponding names is a hallmark of cognitive aging, as is increased susceptibility to distraction. Given evidence that older adults spontaneously encode relationships between target pictures and simultaneously occurring distractors (a hyper-binding phenomenon), we asked whether memory for face-name pairs could be improved through prior exposure to faces presented with distractor names. In three experiments, young and older adults performed a selective attention task on faces while ignoring superimposed names. After a delay, they learned and were tested on face-name pairs that were either maintained or rearranged from the initial task but were not told of the connection between tasks. In each experiment, older but not younger participants showed better memory for maintained than for rearranged pairs, indicating that older adults' natural propensity to tacitly encode and bind relevant and irrelevant information can be employed to aid face-name memory performance.

Isothermal titration calorimetry for measuring macromolecule-ligand affinity.

PubMed

Duff, Michael R; Grubbs, Jordan; Howell, Elizabeth E

2011-09-07

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

PubMed Central

Duff,, Michael R.; Grubbs, Jordan; Howell, Elizabeth E.

2011-01-01

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given. PMID:21931288

Physical and mental effort disrupts the implicit sense of agency.

PubMed

Howard, Emma E; Edwards, S Gareth; Bayliss, Andrew P

2016-12-01

We investigated the effect of effort on implicit agency ascription for actions performed under varying levels of physical effort or cognitive load. People are able to estimate the interval between two events accurately, but they underestimate the interval between their own actions and their outcomes. This effect is known as 'intentional binding', and may provide feedback regarding the consequences of our actions. Concurrently with the interval reproduction task, our participants pulled sports resistance bands at high and low resistance levels (Experiments 1 and 2), or performed a working memory task with high and low set-sizes (Experiment 3). Intentional binding was greater under low than high effort. When the effort was task-related (Experiment 1), this effect depended on the individual's explicit appraisal of exertion, while the effect of effort was evident at the group level when the effort was task-unrelated (physical, Experiment 2; mental, Experiment 3). These findings imply that the process of intentional binding is compromised when cognitive resources are depleted, either through physical or mental strain. We discuss this notion in relation to the integration of direct sensorimotor feedback with signals of agency and other instances of cognitive resource depletion and action control during strain. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhanced Peptide Radiotherapy of Prostate Cancer Using Targeted Adenoviral Vectors

DTIC Science & Technology

2004-06-01

regard to binding of 64Cu - octreotide. In vitro experiments were performed with DU-145 and PC-3 human prostate cancer cells. Expression levels of SSTR2...were determined using a 64Cu -octreotide saturation binding assay on cell membrane preparations. In vivo experiments were conducted in scid mice bearing...subcutaneous DU-l45 or PC-3 cells. AdSSTR2 was injected intratumorally followed 48 h later by an i.v. injection of 64Cu -octreotide. The mice were

Relational and conjunctive binding functions dissociate in short-term memory.

PubMed

Parra, Mario A; Fabi, Katia; Luzzi, Simona; Cubelli, Roberto; Hernandez Valdez, Maria; Della Sala, Sergio

2015-02-01

Remembering complex events requires binding features within unified objects (conjunctions) and holding associations between objects (relations). Recent studies suggest that the two functions dissociate in long-term memory (LTM). Less is known about their functional organization in short-term memory (STM). The present study investigated this issue in patient AE affected by a stroke which caused damage to brain regions known to be relevant for relational functions both in LTM and in STM (i.e., the hippocampus). The assessment involved a battery of standard neuropsychological tasks and STM binding tasks. One STM binding task (Experiment 1) presented common objects and common colors forming either pairs (relations) or integrated objects (conjunctions). Free recall of relations or conjunctions was assessed. A second STM binding task used random polygons and non-primary colors instead (Experiment 2). Memory was assessed by selecting the features that made up the relations or the conjunctions from a set of single polygons and a set of single colors. The neuropsychological assessment revealed impaired delayed memory in AE. AE's pronounced relational STM binding deficits contrasted with his completely preserved conjunctive binding functions in both Experiments 1 and 2. Only 2.35% and 1.14% of the population were expected to have a discrepancy more extreme than that presented by AE in Experiments 1 and 2, respectively. Processing relations and conjunctions of very elementary nonspatial features in STM led to dissociating performances in AE. These findings may inform current theories of memory decline such as those linked to cognitive aging.

Attentional Modulation of Perceptual Comparison for Feature Binding

ERIC Educational Resources Information Center

Kuo, Bo-Cheng; Rotshtein, Pia; Yeh, Yei-Yu

2011-01-01

We investigated the neural correlates of attentional modulation in the perceptual comparison process for detecting feature-binding changes in an event-related functional magnetic resonance imaging (fMRI) experiment. Participants performed a variant of a cued change detection task. They viewed a memory array, a spatial retro-cue, and later a probe…

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

PubMed

Schuller, Marion; Höfner, Georg; Wanner, Klaus T

2017-10-09

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human Lineage-Specific Transcriptional Regulation through GA-Binding Protein Transcription Factor Alpha (GABPa)

PubMed Central

Perdomo-Sabogal, Alvaro; Nowick, Katja; Piccini, Ilaria; Sudbrak, Ralf; Lehrach, Hans; Yaspo, Marie-Laure; Warnatz, Hans-Jörg; Querfurth, Robert

2016-01-01

A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes. PMID:26814189

Binding, Relational Memory, and Recall of Naturalistic Events: A Developmental Perspective

ERIC Educational Resources Information Center

Sluzenski, Julia; Newcombe, Nora S.; Kovacs, Stacie L.

2006-01-01

This research was an investigation of children's performance on a task that requires memory binding. In Experiments 1 and 2, 4-year-olds, 6-year-olds, and adults viewed complex pictures and were tested on memory for isolated parts in the pictures and on the part combinations (combination condition). The results suggested improvement in memory for…

Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

PubMed

Freyberger, Alexius; Wilson, Vickie; Weimer, Marc; Tan, Shirlee; Tran, Hoai-Son; Ahr, Hans-Jürgen

2010-08-01

Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as receptor source from ovariectomized rats, as a recombinant protein is used and thus contributes to the 3R concept (reduce, replace, and refine). Furthermore, in contrast to other assays, this assay could be adjusted to an intermediate/high throughput format. On the whole, this assay is a promising candidate for further validation. Copyright 2010 Elsevier Inc. All rights reserved.

Free Energy Simulations of Ligand Binding to the Aspartate Transporter GltPh

PubMed Central

Heinzelmann, Germano; Baştuğ, Turgut; Kuyucak, Serdar

2011-01-01

Glutamate/Aspartate transporters cotransport three Na+ and one H+ ions with the substrate and countertransport one K+ ion. The binding sites for the substrate and two Na+ ions have been observed in the crystal structure of the archeal homolog GltPh, while the binding site for the third Na+ ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of GltPh, giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na+ ions. They also shed light into Asp/Glu selectivity of GltPh, which is not observed in eukaryotic glutamate transporters. PMID:22098736

Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

PubMed

Freyberger, Alexius; Weimer, Marc; Tran, Hoai-Son; Ahr, Hans-Jürgen

2010-08-01

Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation. Copyright 2009 Elsevier Inc. All rights reserved.

Quantum annealing versus classical machine learning applied to a simplified computational biology problem

PubMed Central

Li, Richard Y.; Di Felice, Rosa; Rohs, Remo; Lidar, Daniel A.

2018-01-01

Transcription factors regulate gene expression, but how these proteins recognize and specifically bind to their DNA targets is still debated. Machine learning models are effective means to reveal interaction mechanisms. Here we studied the ability of a quantum machine learning approach to predict binding specificity. Using simplified datasets of a small number of DNA sequences derived from actual binding affinity experiments, we trained a commercially available quantum annealer to classify and rank transcription factor binding. The results were compared to state-of-the-art classical approaches for the same simplified datasets, including simulated annealing, simulated quantum annealing, multiple linear regression, LASSO, and extreme gradient boosting. Despite technological limitations, we find a slight advantage in classification performance and nearly equal ranking performance using the quantum annealer for these fairly small training data sets. Thus, we propose that quantum annealing might be an effective method to implement machine learning for certain computational biology problems. PMID:29652405

An Effective Approach for Clustering InhA Molecular Dynamics Trajectory Using Substrate-Binding Cavity Features

PubMed Central

Ruiz, Duncan D. A.; Norberto de Souza, Osmar

2015-01-01

Protein receptor conformations, obtained from molecular dynamics (MD) simulations, have become a promising treatment of its explicit flexibility in molecular docking experiments applied to drug discovery and development. However, incorporating the entire ensemble of MD conformations in docking experiments to screen large candidate compound libraries is currently an unfeasible task. Clustering algorithms have been widely used as a means to reduce such ensembles to a manageable size. Most studies investigate different algorithms using pairwise Root-Mean Square Deviation (RMSD) values for all, or part of the MD conformations. Nevertheless, the RMSD only may not be the most appropriate gauge to cluster conformations when the target receptor has a plastic active site, since they are influenced by changes that occur on other parts of the structure. Hence, we have applied two partitioning methods (k-means and k-medoids) and four agglomerative hierarchical methods (Complete linkage, Ward’s, Unweighted Pair Group Method and Weighted Pair Group Method) to analyze and compare the quality of partitions between a data set composed of properties from an enzyme receptor substrate-binding cavity and two data sets created using different RMSD approaches. Ensembles of representative MD conformations were generated by selecting a medoid of each group from all partitions analyzed. We investigated the performance of our new method for evaluating binding conformation of drug candidates to the InhA enzyme, which were performed by cross-docking experiments between a 20 ns MD trajectory and 20 different ligands. Statistical analyses showed that the novel ensemble, which is represented by only 0.48% of the MD conformations, was able to reproduce 75% of all dynamic behaviors within the binding cavity for the docking experiments performed. Moreover, this new approach not only outperforms the other two RMSD-clustering solutions, but it also shows to be a promising strategy to distill biologically relevant information from MD trajectories, especially for docking purposes. PMID:26218832

An Effective Approach for Clustering InhA Molecular Dynamics Trajectory Using Substrate-Binding Cavity Features.

PubMed

De Paris, Renata; Quevedo, Christian V; Ruiz, Duncan D A; Norberto de Souza, Osmar

2015-01-01

Protein receptor conformations, obtained from molecular dynamics (MD) simulations, have become a promising treatment of its explicit flexibility in molecular docking experiments applied to drug discovery and development. However, incorporating the entire ensemble of MD conformations in docking experiments to screen large candidate compound libraries is currently an unfeasible task. Clustering algorithms have been widely used as a means to reduce such ensembles to a manageable size. Most studies investigate different algorithms using pairwise Root-Mean Square Deviation (RMSD) values for all, or part of the MD conformations. Nevertheless, the RMSD only may not be the most appropriate gauge to cluster conformations when the target receptor has a plastic active site, since they are influenced by changes that occur on other parts of the structure. Hence, we have applied two partitioning methods (k-means and k-medoids) and four agglomerative hierarchical methods (Complete linkage, Ward's, Unweighted Pair Group Method and Weighted Pair Group Method) to analyze and compare the quality of partitions between a data set composed of properties from an enzyme receptor substrate-binding cavity and two data sets created using different RMSD approaches. Ensembles of representative MD conformations were generated by selecting a medoid of each group from all partitions analyzed. We investigated the performance of our new method for evaluating binding conformation of drug candidates to the InhA enzyme, which were performed by cross-docking experiments between a 20 ns MD trajectory and 20 different ligands. Statistical analyses showed that the novel ensemble, which is represented by only 0.48% of the MD conformations, was able to reproduce 75% of all dynamic behaviors within the binding cavity for the docking experiments performed. Moreover, this new approach not only outperforms the other two RMSD-clustering solutions, but it also shows to be a promising strategy to distill biologically relevant information from MD trajectories, especially for docking purposes.

A Course-Based Undergraduate Research Experience Investigating p300 Bromodomain Mutations#

PubMed Central

Shanle, Erin K.; Tsun, Ian K.; Strahl, Brian D.

2016-01-01

Course-based undergraduate research experiences (CUREs) provide an opportunity for students to engage in experiments with outcomes that are unknown to both the instructor and students. These experiences allow students and instructors to collaboratively bridge the research laboratory and classroom, and provide research experiences for a large number of students relative to traditional individual mentored research. Here, we describe a molecular biology CURE investigating the impact of clinically relevant mutations found in the bromodomain of the p300 transcriptional regulator on acetylated histone interaction. In the CURE, students identified missense mutations in the p300 bromo-domain using the Catalogue of Somatic Mutations in Cancer (COSMIC) database and hypothesized the effects of the mutation on the acetyl-binding function of the domain. They cloned and purified the mutated bromodomain and performed peptide pulldown assays to define its potential to bind to acetylated histones. Upon completion of the course, students showed increased confidence performing molecular techniques and reported positively on doing a research project in class. In addition, results generated in the classroom were further validated in the research laboratory setting thereby providing a new model for faculty to engage in both course-based and individual undergraduate research experiences. PMID:26537758

Binding and strategic selection in working memory: a lifespan dissociation.

PubMed

Sander, Myriam C; Werkle-Bergner, Markus; Lindenberger, Ulman

2011-09-01

Working memory (WM) shows a gradual increase during childhood, followed by accelerating decline from adulthood to old age. To examine these lifespan differences more closely, we asked 34 children (10-12 years), 40 younger adults (20-25 years), and 39 older adults (70-75 years) to perform a color change detection task. Load levels and encoding durations were varied for displays including targets only (Experiment 1) or targets plus distracters (Experiment 2, investigating a subsample of Experiment 1). WM performance was lower in older adults and children than in younger adults. Longer presentation times were associated with better performance in all age groups, presumably reflecting increasing effects of strategic selection mechanisms on WM performance. Children outperformed older adults when encoding times were short, and distracter effects were larger in children and older adults than in younger adults. We conclude that strategic selection in WM develops more slowly during childhood than basic binding operations, presumably reflecting the delay in maturation of frontal versus medio-temporal brain networks. In old age, both sets of mechanisms decline, reflecting senescent change in both networks. We discuss similarities to episodic memory development and address open questions for future research.

Short-term memory binding deficits in Alzheimer's disease.

PubMed

Parra, Mario A; Abrahams, Sharon; Fabi, Katia; Logie, Robert; Luzzi, Simona; Della Sala, Sergio

2009-04-01

Alzheimer's disease impairs long term memories for related events (e.g. faces with names) more than for single events (e.g. list of faces or names). Whether or not this associative or 'binding' deficit is also found in short-term memory has not yet been explored. In two experiments we investigated binding deficits in verbal short-term memory in Alzheimer's disease. Experiment 1: 23 patients with Alzheimer's disease and 23 age and education matched healthy elderly were recruited. Participants studied visual arrays of objects (six for healthy elderly and four for Alzheimer's disease patients), colours (six for healthy elderly and four for Alzheimer's disease patients), unbound objects and colours (three for healthy elderly and two for Alzheimer's disease patients in each of the two categories), or objects bound with colours (three for healthy elderly and two for Alzheimer's disease patients). They were then asked to recall the items verbally. The memory of patients with Alzheimer's disease for objects bound with colours was significantly worse than for single or unbound features whereas healthy elderly's memory for bound and unbound features did not differ. Experiment 2: 21 Alzheimer's disease patients and 20 matched healthy elderly were recruited. Memory load was increased for the healthy elderly group to eight items in the conditions assessing memory for single or unbound features and to four items in the condition assessing memory for the binding of these features. For Alzheimer's disease patients the task remained the same. This manipulation permitted the performance to be equated across groups in the conditions assessing memory for single or unbound features. The impairment in Alzheimer's disease patients in recalling bound objects reported in Experiment 1 was replicated. The binding cost was greater than that observed in the healthy elderly group, who did not differ in their performance for bound and unbound features. Alzheimer's disease grossly impairs the mechanisms responsible for holding integrated objects in verbal short-term memory.

Retrieval from long-term memory reduces working memory representations for visual features and their bindings.

PubMed

van Lamsweerde, Amanda E; Beck, Melissa R; Elliott, Emily M

2015-02-01

The ability to remember feature bindings is an important measure of the ability to maintain objects in working memory (WM). In this study, we investigated whether both object- and feature-based representations are maintained in WM. Specifically, we tested the hypotheses that retaining a greater number of feature representations (i.e., both as individual features and bound representations) results in a more robust representation of individual features than of feature bindings, and that retrieving information from long-term memory (LTM) into WM would cause a greater disruption to feature bindings. In four experiments, we examined the effects of retrieving a word from LTM on shape and color-shape binding change detection performance. We found that binding changes were more difficult to detect than individual-feature changes overall, but that the cost of retrieving a word from LTM was the same for both individual-feature and binding changes.

Characterization of lithium coordination sites with magic-angle spinning NMR

NASA Astrophysics Data System (ADS)

Haimovich, A.; Goldbourt, A.

2015-05-01

Lithium, in the form of lithium carbonate, is one of the most common drugs for bipolar disorder. Lithium is also considered to have an effect on many other cellular processes hence it possesses additional therapeutic as well as side effects. In order to quantitatively characterize the binding mode of lithium, it is required to identify the interacting species and measure their distances from the metal center. Here we use magic-angle spinning (MAS) solid-state NMR to study the binding site of lithium in complex with glycine and water (LiGlyW). Such a compound is a good enzyme mimetic since lithium is four-coordinated to one water molecule and three carboxylic groups. Distance measurements to carbons are performed using a 2D transferred echo double resonance (TEDOR) MAS solid-state NMR experiment, and water binding is probed by heteronuclear high-resolution proton-lithium and proton-carbon correlation (wPMLG-HETCOR) experiments. Both HETCOR experiments separate the main complex from impurities and non-specifically bound lithium species, demonstrating the sensitivity of the method to probe the species in the binding site. Optimizations of the TEDOR pulse scheme in the case of a quadrupolar nucleus with a small quadrupole coupling constant show that it is most efficient when pulses are positioned on the spin-1/2 (carbon-13) nucleus. Since the intensity of the TEDOR signal is not normalized, careful data analysis that considers both intensity and dipolar oscillations has to be performed. Nevertheless we show that accurate distances can be extracted for both carbons of the bound glycine and that these distances are consistent with the X-ray data and with lithium in a tetrahedral environment. The lithium environment in the complex is very similar to the binding site in inositol monophosphatase, an enzyme associated with bipolar disorder and the putative target for lithium therapy. A 2D TEDOR experiment applied to the bacterial SuhB gene product of this enzyme was designed to probe direct correlations between lithium, the enzyme inhibitor, and the closest carboxyl carbons of the binding site. At this point, the chemical shift of the bound carboxyl groups in this 29 kDa enzyme could be determined.

Lack of color integration in visual short-term memory binding.

PubMed

Parra, Mario A; Cubelli, Roberto; Della Sala, Sergio

2011-10-01

Bicolored objects are retained in visual short-term memory (VSTM) less efficiently than unicolored objects. This is unlike shape-color combinations, whose retention in VSTM does not differ from that observed for shapes only. It is debated whether this is due to a lack of color integration and whether this may reflect the function of separate memory mechanisms. Participants judged whether the colors of bicolored objects (each with an external and an internalcolor) were the same or different across two consecutive screens. Colors had to be remembered either individually or in combination. In Experiment 1, external colors in the combined colors condition were remembered better than the internal colors, and performance for both was worse than that in the individual colors condition. The lack of color integration observed in Experiment 1 was further supported by a reduced capacity of VSTM to retain color combinations, relative to individual colors (Experiment 2). An additional account was found in Experiment 3, which showed spared color-color binding in the presence of impaired shape-color binding in a brain-damaged patient, thus suggesting that these two memory mechanisms are different.

Regulation of the Myxococcus xanthus C-Signal-Dependent Ω4400 Promoter by the Essential Developmental Protein FruA

PubMed Central

Yoder-Himes, Deborah R.; Kroos, Lee

2006-01-01

The bacterium Myxococcus xanthus employs extracellular signals to coordinate aggregation and sporulation during multicellular development. Extracellular, contact-dependent signaling that involves the CsgA protein (called C-signaling) activates FruA, a putative response regulator that governs a branched signaling pathway inside cells. One branch regulates cell movement, leading to aggregation. The other branch regulates gene expression, leading to sporulation. C-signaling is required for full expression of most genes induced after 6 h into development, including the gene identified by Tn5 lac insertion Ω4400. To determine if FruA is a direct regulator of Ω4400 transcription, a combination of in vivo and in vitro experiments was performed. Ω4400 expression was abolished in a fruA mutant. The DNA-binding domain of FruA bound specifically to DNA upstream of the promoter −35 region in vitro. Mutations between bp −86 and −77 greatly reduced binding. One of these mutations had been shown previously to reduce Ω4400 expression in vivo and make it independent of C-signaling. For the first time, chromatin immunoprecipitation (ChIP) experiments were performed on M. xanthus. The ChIP experiments demonstrated that FruA is associated with the Ω4400 promoter region late in development, even in the absence of C-signaling. Based on these results, we propose that FruA directly activates Ω4400 transcription to a moderate level prior to C-signaling and, in response to C-signaling, binds near bp −80 and activates transcription to a higher level. Also, the highly localized effects of mutations between bp −86 and −77 on DNA binding in vitro, together with recently published footprints, allow us to predict a consensus binding site of GTCG/CGA/G for the FruA DNA-binding domain. PMID:16816188

Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module*

PubMed Central

Taylor, Courtney B.; Talib, M. Faiz; McCabe, Clare; Bu, Lintao; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3–6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function. PMID:22147693

Low-Quality Structural and Interaction Data Improves Binding Affinity Prediction via Random Forest.

PubMed

Li, Hongjian; Leung, Kwong-Sak; Wong, Man-Hon; Ballester, Pedro J

2015-06-12

Docking scoring functions can be used to predict the strength of protein-ligand binding. It is widely believed that training a scoring function with low-quality data is detrimental for its predictive performance. Nevertheless, there is a surprising lack of systematic validation experiments in support of this hypothesis. In this study, we investigated to which extent training a scoring function with data containing low-quality structural and binding data is detrimental for predictive performance. We actually found that low-quality data is not only non-detrimental, but beneficial for the predictive performance of machine-learning scoring functions, though the improvement is less important than that coming from high-quality data. Furthermore, we observed that classical scoring functions are not able to effectively exploit data beyond an early threshold, regardless of its quality. This demonstrates that exploiting a larger data volume is more important for the performance of machine-learning scoring functions than restricting to a smaller set of higher data quality.

Camouflaging endothelial cells: does it prolong graft survival?

PubMed

Stuhlmeier, K M; Lin, Y

1999-08-05

Camouflaging antigens on the surface of cells seems an appealing way to prevent activation of the immune system. We explored the possibility of preventing hyperacute rejection by chemically camouflaging endothelial cells (EC). In vitro as well as in vivo experiments were performed. First, the ability of mPEG coating to prevent antibody-antigen interactions was evaluated. Second, we tested the degree to which mPEG coating prevents activation of EC by stimuli such as TNF-alpha and LPS. Third, in vivo experiments were performed to test the ability of mPEG coating to prolong xenograft survival. We demonstrate that binding of several antibodies to EC or serum proteins can be inhibited by mPEG. Furthermore, binding of TNF-alpha as well as LPS to EC is blocked since mPEG treatment of EC inhibits the subsequent up-regulation of E-selectin by these stimuli. However, in vivo experiments revealed that currently this method alone is not sufficient to prevent hyperacute rejection.

Combining in silico and in cerebro approaches for virtual screening and pose prediction in SAMPL4.

PubMed

Voet, Arnout R D; Kumar, Ashutosh; Berenger, Francois; Zhang, Kam Y J

2014-04-01

The SAMPL challenges provide an ideal opportunity for unbiased evaluation and comparison of different approaches used in computational drug design. During the fourth round of this SAMPL challenge, we participated in the virtual screening and binding pose prediction on inhibitors targeting the HIV-1 integrase enzyme. For virtual screening, we used well known and widely used in silico methods combined with personal in cerebro insights and experience. Regular docking only performed slightly better than random selection, but the performance was significantly improved upon incorporation of additional filters based on pharmacophore queries and electrostatic similarities. The best performance was achieved when logical selection was added. For the pose prediction, we utilized a similar consensus approach that amalgamated the results of the Glide-XP docking with structural knowledge and rescoring. The pose prediction results revealed that docking displayed reasonable performance in predicting the binding poses. However, prediction performance can be improved utilizing scientific experience and rescoring approaches. In both the virtual screening and pose prediction challenges, the top performance was achieved by our approaches. Here we describe the methods and strategies used in our approaches and discuss the rationale of their performances.

Combining in silico and in cerebro approaches for virtual screening and pose prediction in SAMPL4

NASA Astrophysics Data System (ADS)

Voet, Arnout R. D.; Kumar, Ashutosh; Berenger, Francois; Zhang, Kam Y. J.

2014-04-01

The SAMPL challenges provide an ideal opportunity for unbiased evaluation and comparison of different approaches used in computational drug design. During the fourth round of this SAMPL challenge, we participated in the virtual screening and binding pose prediction on inhibitors targeting the HIV-1 integrase enzyme. For virtual screening, we used well known and widely used in silico methods combined with personal in cerebro insights and experience. Regular docking only performed slightly better than random selection, but the performance was significantly improved upon incorporation of additional filters based on pharmacophore queries and electrostatic similarities. The best performance was achieved when logical selection was added. For the pose prediction, we utilized a similar consensus approach that amalgamated the results of the Glide-XP docking with structural knowledge and rescoring. The pose prediction results revealed that docking displayed reasonable performance in predicting the binding poses. However, prediction performance can be improved utilizing scientific experience and rescoring approaches. In both the virtual screening and pose prediction challenges, the top performance was achieved by our approaches. Here we describe the methods and strategies used in our approaches and discuss the rationale of their performances.

Specific deficit of colour-colour short-term memory binding in sporadic and familial Alzheimer's disease.

PubMed

Parra, Mario A; Sala, Sergio Della; Abrahams, Sharon; Logie, Robert H; Méndez, Luis Guillermo; Lopera, Francisco

2011-06-01

Short-term memory binding of visual features which are processed across different dimensions (shape-colour) is impaired in sporadic Alzheimer's disease, familial Alzheimer's disease, and in asymptomatic carriers of familial Alzheimer's disease. This study investigated whether Alzheimer's disease also impacts on within-dimension binding processes. The study specifically explored whether visual short-term memory binding of features of the same type (colour-colour) is sensitive to Alzheimer's disease. We used a neuropsychological battery and a short-term memory binding task to assess patients with sporadic Alzheimer's disease (Experiment 1), familial Alzheimer's disease (Experiment 2) due to the mutation E280A of the Presenilin-1 gene and asymptomatic carriers of the mutation. The binding task assessed change detection within arrays of unicoloured objects (Colour Only) or bicoloured objects the colours of which had to be remembered separately (Unbound Colours) or together (Bound Colours). Performance on the Bound Colours condition (1) explained the largest proportion of variance between patients (sporadic and familial Alzheimer's disease), (2) combined more sensitivity and specificity for the disease than other more traditional neuropsychological tasks, (3) identified asymptomatic carriers of the mutation even when traditional neuropsychological measures and other measures of short-term memory did not and, (4) contrary to shape-colour binding, correlated with measures of hippocampal functions. Colour-colour binding and shape-colour binding both appear to be sensitive to AD even though they seem to rely on different brain mechanisms. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

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

Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is notmore » involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.« less

Characterization of strychnine-sensitive glycine receptor in the intact frog retina: modulation by protein kinases.

PubMed

Salceda, Rocío; Aguirre-Ramirez, Marisela

2005-03-01

We studied 3H-glycine and 3H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a KD of 2.5 and 2.0 microM, respectively. Specific binding of glycine was displaced by beta-alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.

Enantioselective binding of L, D-phenylalanine to ct DNA

NASA Astrophysics Data System (ADS)

Zhang, Lijin; Xu, Jianhua; Huang, Yan; Min, Shungeng

2009-10-01

The enantioselective binding of L, D-phenylalanine to calf thymus DNA was studied by absorption, circular dichroism, fluorescence quenching, viscosity, salt effect and emission experiments. The results obtained from absorption, circular dichroism, fluorescence quenching and viscosity experiments excluded the intercalative binding and salt effect experiments did not support electrostatic binding. So the binding of L, D-phenylalanine to ct DNA should be groove binding. Furthermore, the emission spectra revealed that the binding is enantioselective.

Enantioselective binding of L,D-phenylalanine to ct DNA.

PubMed

Zhang, Lijin; Xu, Jianhua; Huang, Yan; Min, Shungeng

2009-10-15

The enantioselective binding of L,D-phenylalanine to calf thymus DNA was studied by absorption, circular dichroism, fluorescence quenching, viscosity, salt effect and emission experiments. The results obtained from absorption, circular dichroism, fluorescence quenching and viscosity experiments excluded the intercalative binding and salt effect experiments did not support electrostatic binding. So the binding of l,d-phenylalanine to ct DNA should be groove binding. Furthermore, the emission spectra revealed that the binding is enantioselective.

The crystal structure of the AgamOBP1•Icaridin complex reveals alternative binding modes and stereo-selective repellent recognition.

PubMed

Drakou, Christina E; Tsitsanou, Katerina E; Potamitis, Constantinos; Fessas, Dimitrios; Zervou, Maria; Zographos, Spyros E

2017-01-01

Anopheles gambiae Odorant Binding Protein 1 in complex with the most widely used insect repellent DEET, was the first reported crystal structure of an olfactory macromolecule with a repellent, and paved the way for OBP1-structure-based approaches for discovery of new host-seeking disruptors. In this work, we performed STD-NMR experiments to directly monitor and verify the formation of a complex between AgamOBP1 and Icaridin, an efficient DEET alternative. Furthermore, Isothermal Titration Calorimetry experiments provided evidence for two Icaridin-binding sites with different affinities (Kd = 0.034 and 0.714 mM) and thermodynamic profiles of ligand binding. To elucidate the binding mode of Icaridin, the crystal structure of AgamOBP1•Icaridin complex was determined at 1.75 Å resolution. We found that Icaridin binds to the DEET-binding site in two distinct orientations and also to a novel binding site located at the C-terminal region. Importantly, only the most active 1R,2S-isomer of Icaridin's equimolar diastereoisomeric mixture binds to the AgamOBP1 crystal, providing structural evidence for the possible contribution of OBP1 to the stereoselectivity of Icaridin perception in mosquitoes. Structural analysis revealed two ensembles of conformations differing mainly in spatial arrangement of their sec-butyl moieties. Moreover, structural comparison with DEET indicates a common recognition mechanism for these structurally related repellents. Ligand interactions with both sites and binding modes were further confirmed by 2D 1 H- 15 N HSQC NMR spectroscopy. The identification of a novel repellent-binding site in AgamOBP1 and the observed structural conservation and stereoselectivity of its DEET/Icaridin-binding sites open new perspectives for the OBP1-structure-based discovery of next-generation insect repellents.

Molecular simulations of multimodal ligand-protein binding: elucidation of binding sites and correlation with experiments.

PubMed

Freed, Alexander S; Garde, Shekhar; Cramer, Steven M

2011-11-17

Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. MD simulations also identify additional binding sites that were not observed in the NMR experiments. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.

A common perceptual temporal limit of binding synchronous inputs across different sensory attributes and modalities.

PubMed

Fujisaki, Waka; Nishida, Shin'ya

2010-08-07

The human brain processes different aspects of the surrounding environment through multiple sensory modalities, and each modality can be subdivided into multiple attribute-specific channels. When the brain rebinds sensory content information ('what') across different channels, temporal coincidence ('when') along with spatial coincidence ('where') provides a critical clue. It however remains unknown whether neural mechanisms for binding synchronous attributes are specific to each attribute combination, or universal and central. In human psychophysical experiments, we examined how combinations of visual, auditory and tactile attributes affect the temporal frequency limit of synchrony-based binding. The results indicated that the upper limits of cross-attribute binding were lower than those of within-attribute binding, and surprisingly similar for any combination of visual, auditory and tactile attributes (2-3 Hz). They are unlikely to be the limits for judging synchrony, since the temporal limit of a cross-attribute synchrony judgement was higher and varied with the modality combination (4-9 Hz). These findings suggest that cross-attribute temporal binding is mediated by a slow central process that combines separately processed 'what' and 'when' properties of a single event. While the synchrony performance reflects temporal bottlenecks existing in 'when' processing, the binding performance reflects the central temporal limit of integrating 'when' and 'what' properties.

Identical linkage and cooperativity of oxygen and carbon monoxide binding to Octopus dofleini hemocyanin

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

Connelly, P.R.; Gill, S.J.; Miller, K.I.

1989-02-21

Employment of high-precision thin-layer methods has enabled detailed functional characterization of oxygen and carbon monoxide binding for (1) the fully assembled form with 70 binding sites and (2) the isolated chains with 7 binding sites of octopus dofleini hemocyanin. The striking difference in the cooperativities of the two ligands for the assembled decamer is revealed through an examination of the binding capacities and the partition coefficient, determined as functions of the activities of both ligands. A global analysis of the data sets supported by a two-state allosteric model assuming an allosteric unit of 7. Higher level allosteric interactions were notmore » indicated. This contrasts to results obtained for arthropod hemocyanins. Oxygen and carbon monoxide experiments performed on the isolated subunit chain confirmed the presence of functional heterogeneity reported previously. The analysis shows two types of binding sites in the ratio of 4:3.« less

Characterization of interaction between doxycycline and human serum albumin by capillary electrophoresis-frontal analysis.

PubMed

Sun, Hanwen; He, Pan

2009-06-01

The binding of doxycycline to HSA under simulated physiological conditions (pH 7.4, 67 mM phosphate, I=0.17, drug concentration 100 microM, HSA concentration up to 475 microM, 36.5 degrees C) was studied by CE-frontal analysis. The number of primary binding sites, binding constant and physiological protein-binding percentage were 1.9, 1.51 x 10(3) M(-1) and 59.80%, respectively. In addition, the thermodynamic parameters including enthalpy change (DeltaH), entropy change (DeltaS) and free energy change (DeltaG) of the reaction were obtained in order to characterize the acting forces between doxycycline and HSA. Furthermore, to better understand the nature of doxycycline-HSA binding and to get information about potential interaction with other drugs, displacement experiments were performed. The results showed that doxycycline binds at site II of HSA.

Development of peptoid-based ligands for the removal of cadmium from biological media

DOE PAGES

Knight, Abigail S.; Zhou, Effie Y.; Francis, Matthew B.

2015-05-14

Cadmium poisoning poses a serious health concern due to cadmium's increasing industrial use, yet there is currently no recommended treatment. The selective coordination of cadmium in a biological environment—i.e. in the presence of serum ions, small molecules, and proteins—is a difficult task. To address this challenge, a combinatorial library of peptoid-based ligands has been evaluated to identify structures that selectively bind to cadmium in human serum with minimal chelation of essential metal ions. Eighteen unique ligands were identified in this screening procedure, and the binding affinity of each was measured using metal titrations monitored by UV-vis spectroscopy. To evaluate themore » significance of each chelating moiety, sequence rearrangements and substitutions were examined. Analysis of a metal–ligand complex by NMR spectroscopy highlighted the importance of particular residues. Depletion experiments were performed in serum mimetics and human serum with exogenously added cadmium. These depletion experiments were used to compare and demonstrate the ability of these peptoids to remove cadmium from blood-like mixtures. In one of these depletion experiments, the peptoid sequence was able to deplete the cadmium to a level comparable to the reported acute toxicity limit. Evaluation of the metal selectivity in buffered solution and in human serum was performed to verify minimal off-target binding. These studies highlight a screening platform for the identification of metal–ligands that are capable of binding in a complex environment. They additionally demonstrate the potential utility of biologically-compatible ligands for the treatment of heavy metal poisoning.« less

Development of peptoid-based ligands for the removal of cadmium from biological media

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

Knight, Abigail S.; Zhou, Effie Y.; Francis, Matthew B.

Cadmium poisoning poses a serious health concern due to cadmium's increasing industrial use, yet there is currently no recommended treatment. The selective coordination of cadmium in a biological environment—i.e. in the presence of serum ions, small molecules, and proteins—is a difficult task. To address this challenge, a combinatorial library of peptoid-based ligands has been evaluated to identify structures that selectively bind to cadmium in human serum with minimal chelation of essential metal ions. Eighteen unique ligands were identified in this screening procedure, and the binding affinity of each was measured using metal titrations monitored by UV-vis spectroscopy. To evaluate themore » significance of each chelating moiety, sequence rearrangements and substitutions were examined. Analysis of a metal–ligand complex by NMR spectroscopy highlighted the importance of particular residues. Depletion experiments were performed in serum mimetics and human serum with exogenously added cadmium. These depletion experiments were used to compare and demonstrate the ability of these peptoids to remove cadmium from blood-like mixtures. In one of these depletion experiments, the peptoid sequence was able to deplete the cadmium to a level comparable to the reported acute toxicity limit. Evaluation of the metal selectivity in buffered solution and in human serum was performed to verify minimal off-target binding. These studies highlight a screening platform for the identification of metal–ligands that are capable of binding in a complex environment. They additionally demonstrate the potential utility of biologically-compatible ligands for the treatment of heavy metal poisoning.« less

The crystal structure of ribonuclease A in complex with thymidine-3'-monophosphate provides further insight into ligand binding.

PubMed

Doucet, Nicolas; Jayasundera, Thusitha B; Simonović, Miljan; Loria, J Patrick

2010-08-15

Thymidine-3'-monophosphate (3'-TMP) is a competitive inhibitor analogue of the 3'-CMP and 3'-UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3'-TMP binds the enzyme with a dissociation constant (K(d)) of 15 microM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3'-TMP at 1.55 A resolution and we have performed NMR binding experiments with 3'-CMP and 3'-TMP. Our results show that binding of 3'-TMP is very similar to other natural and non-natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2'-hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme. 2010 Wiley-Liss, Inc.

A Lysine at the C-Terminus of an Odorant-Binding Protein is Involved in Binding Aldehyde Pheromone Components in Two Helicoverpa Species

PubMed Central

Sun, Ya-Lan; Huang, Ling-Qiao; Pelosi, Paolo; Wang, Chen-Zhu

2013-01-01

Odorant-binding proteins (OBPs) are soluble proteins, whose role in olfaction of insects is being recognized as more and more important. We have cloned, expressed and purified an OBP (HarmOBP7) from the antennae of the moth Helicoverpa armigera. Western blot experiments indicate specific expression of this protein in the antennae of adults. HarmOBP7 binds both pheromone components Z-11-hexadecenal and Z-9-hexadecenal with good affinity. We have also performed a series of binding experiments with linear aldehydes, alcohols and esters, as well as with other compounds and found a requirement of medium size for best affinity. The affinity of OBP7, as well as that of a mutant lacking the last 6 residues does not substantially decrease in acidic conditions, but increases at basic pH values with no significant differences between wild-type and mutant. Binding to both pheromone components, instead, is negatively affected by the lack of the C-terminus. A second mutant, where one of the three lysine residues in the C-terminus (Lys123) was replaced by methionine showed reduced affinity to both pheromone components, as well as to their analogues, thus indicating that Lys123 is involved in binding these compounds, likely forming hydrogen bonds with the functional groups of the ligands. PMID:23372826

Memory Systems Do Not Divide on Consciousness: Reinterpreting Memory in Terms of Activation and Binding

ERIC Educational Resources Information Center

Reder, Lynne M.; Park, Heekyeong; Kieffaber, Paul D.

2009-01-01

There is a popular hypothesis that performance on implicit and explicit memory tasks reflects 2 distinct memory systems. Explicit memory is said to store those experiences that can be consciously recollected, and implicit memory is said to store experiences and affect subsequent behavior but to be unavailable to conscious awareness. Although this…

A course-based undergraduate research experience investigating p300 bromodomain mutations.

PubMed

Shanle, Erin K; Tsun, Ian K; Strahl, Brian D

2016-01-01

Course-based undergraduate research experiences (CUREs) provide an opportunity for students to engage in experiments with outcomes that are unknown to both the instructor and students. These experiences allow students and instructors to collaboratively bridge the research laboratory and classroom, and provide research experiences for a large number of students relative to traditional individual mentored research. Here, we describe a molecular biology CURE investigating the impact of clinically relevant mutations found in the bromodomain of the p300 transcriptional regulator on acetylated histone interaction. In the CURE, students identified missense mutations in the p300 bromodomain using the Catalogue of Somatic Mutations in Cancer (COSMIC) database and hypothesized the effects of the mutation on the acetyl-binding function of the domain. They cloned and purified the mutated bromodomain and performed peptide pulldown assays to define its potential to bind to acetylated histones. Upon completion of the course, students showed increased confidence performing molecular techniques and reported positively on doing a research project in class. In addition, results generated in the classroom were further validated in the research laboratory setting thereby providing a new model for faculty to engage in both course-based and individual undergraduate research experiences. © 2015 The International Union of Biochemistry and Molecular Biology.

The role of aging in intra-item and item-context binding processes in visual working memory.

PubMed

Peterson, Dwight J; Naveh-Benjamin, Moshe

2016-11-01

Aging is accompanied by declines in both working memory and long-term episodic memory processes. Specifically, important age-related memory deficits are characterized by performance impairments exhibited by older relative to younger adults when binding distinct components into a single integrated representation, despite relatively intact memory for the individual components. While robust patterns of age-related binding deficits are prevalent in studies of long-term episodic memory, observations of such deficits in visual working memory (VWM) may depend on the specific type of binding process being examined. For instance, a number of studies indicate that processes involved in item-context binding of items to occupied spatial locations within visual working memory are impaired in older relative to younger adults. Other findings suggest that intra-item binding of visual surface features (e.g., color, shape), compared to memory for single features, within visual working memory, remains relatively intact. Here, we examined each of these binding processes in younger and older adults under both optimal conditions (i.e., no concurrent load) and concurrent load (e.g., articulatory suppression, backward counting). Experiment 1 revealed an age-related intra-item binding deficit for surface features under no concurrent load but not when articulatory suppression was required. In contrast, in Experiments 2 and 3, we observed an age-related item-context binding deficit regardless of the level of concurrent load. These findings reveal that the influence of concurrent load on distinct binding processes within VWM, potentially those supported by rehearsal, is an important factor mediating the presence or absence of age-related binding deficits within VWM. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

PubMed

Werle, E; Lenz, T; Strobel, G; Weicker, H

1988-07-01

The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off

Quantum annealing versus classical machine learning applied to a simplified computational biology problem

NASA Astrophysics Data System (ADS)

Li, Richard Y.; Di Felice, Rosa; Rohs, Remo; Lidar, Daniel A.

2018-03-01

Transcription factors regulate gene expression, but how these proteins recognize and specifically bind to their DNA targets is still debated. Machine learning models are effective means to reveal interaction mechanisms. Here we studied the ability of a quantum machine learning approach to classify and rank binding affinities. Using simplified data sets of a small number of DNA sequences derived from actual binding affinity experiments, we trained a commercially available quantum annealer to classify and rank transcription factor binding. The results were compared to state-of-the-art classical approaches for the same simplified data sets, including simulated annealing, simulated quantum annealing, multiple linear regression, LASSO, and extreme gradient boosting. Despite technological limitations, we find a slight advantage in classification performance and nearly equal ranking performance using the quantum annealer for these fairly small training data sets. Thus, we propose that quantum annealing might be an effective method to implement machine learning for certain computational biology problems.

G =  MAT: linking transcription factor expression and DNA binding data.

PubMed

Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

2011-01-31

Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/.

G = MAT: Linking Transcription Factor Expression and DNA Binding Data

PubMed Central

Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

2011-01-01

Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/. PMID:21297945

Executive Resources and Item-Context Binding: Exploring the Influence of Concurrent Inhibition, Updating, and Shifting Tasks on Context Memory

PubMed Central

Nieznański, Marek; Obidziński, Michał; Zyskowska, Emilia; Niedziałkowska, Daria

2015-01-01

Previous research has demonstrated that context memory performance decreases as a result of cognitive load. However, the role of specific executive resources availability has not been specified yet. In a dual-task experiment, participants performed three kinds of concurrent task engaging: inhibition, updating, or shifting operations. In comparison with a no-load single-task condition, a significant decrease in item and context memory was observed, regardless of the kind of executive task. When executive load conditions were compared with non-specific cognitive load conditions, a significant interference effect was observed in the case of the inhibition task. The inhibition process appears to be an aspect of executive control, which relies on the same resource as item-context binding does, especially when binding refers to associations retrieved from long-term memory. PMID:26435761

Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery.

PubMed

Patching, Simon G

2014-01-01

Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique for the study of ligand binding interactions with membrane proteins, which are the major molecular targets for validated drugs and for current and foreseeable drug discovery. SPR is label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligand molecules using relatively small quantities of materials and has potential to be medium-throughput. The conventional SPR technique requires one binding component to be immobilised on a sensor chip whilst the other binding component in solution is flowed over the sensor surface; a binding interaction is detected using an optical method that measures small changes in refractive index at the sensor surface. This review first describes the basic SPR experiment and the challenges that have to be considered for performing SPR experiments that measure membrane protein-ligand binding interactions, most importantly having the membrane protein in a lipid or detergent environment that retains its native structure and activity. It then describes a wide-range of membrane protein systems for which ligand binding interactions have been characterised using SPR, including the major drug targets G protein-coupled receptors, and how challenges have been overcome for achieving this. Finally it describes some recent advances in SPR-based technology and future potential of the technique to screen ligand binding in the discovery of drugs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding. Copyright © 2013 Elsevier B.V. All rights reserved.

RNA-protein binding motifs mining with a new hybrid deep learning based cross-domain knowledge integration approach.

PubMed

Pan, Xiaoyong; Shen, Hong-Bin

2017-02-28

RNAs play key roles in cells through the interactions with proteins known as the RNA-binding proteins (RBP) and their binding motifs enable crucial understanding of the post-transcriptional regulation of RNAs. How the RBPs correctly recognize the target RNAs and why they bind specific positions is still far from clear. Machine learning-based algorithms are widely acknowledged to be capable of speeding up this process. Although many automatic tools have been developed to predict the RNA-protein binding sites from the rapidly growing multi-resource data, e.g. sequence, structure, their domain specific features and formats have posed significant computational challenges. One of current difficulties is that the cross-source shared common knowledge is at a higher abstraction level beyond the observed data, resulting in a low efficiency of direct integration of observed data across domains. The other difficulty is how to interpret the prediction results. Existing approaches tend to terminate after outputting the potential discrete binding sites on the sequences, but how to assemble them into the meaningful binding motifs is a topic worth of further investigation. In viewing of these challenges, we propose a deep learning-based framework (iDeep) by using a novel hybrid convolutional neural network and deep belief network to predict the RBP interaction sites and motifs on RNAs. This new protocol is featured by transforming the original observed data into a high-level abstraction feature space using multiple layers of learning blocks, where the shared representations across different domains are integrated. To validate our iDeep method, we performed experiments on 31 large-scale CLIP-seq datasets, and our results show that by integrating multiple sources of data, the average AUC can be improved by 8% compared to the best single-source-based predictor; and through cross-domain knowledge integration at an abstraction level, it outperforms the state-of-the-art predictors by 6%. Besides the overall enhanced prediction performance, the convolutional neural network module embedded in iDeep is also able to automatically capture the interpretable binding motifs for RBPs. Large-scale experiments demonstrate that these mined binding motifs agree well with the experimentally verified results, suggesting iDeep is a promising approach in the real-world applications. The iDeep framework not only can achieve promising performance than the state-of-the-art predictors, but also easily capture interpretable binding motifs. iDeep is available at http://www.csbio.sjtu.edu.cn/bioinf/iDeep.

Humic Acid Complexation of Th, Hf and Zr in Ligand Competition Experiments: Metal Loading and Ph Effects

NASA Technical Reports Server (NTRS)

Stern, Jennifer C.; Foustoukos, Dionysis I.; Sonke, Jeroen E.; Salters, Vincent J. M.

2014-01-01

The mobility of metals in soils and subsurface aquifers is strongly affected by sorption and complexation with dissolved organic matter, oxyhydroxides, clay minerals, and inorganic ligands. Humic substances (HS) are organic macromolecules with functional groups that have a strong affinity for binding metals, such as actinides. Thorium, often studied as an analog for tetravalent actinides, has also been shown to strongly associate with dissolved and colloidal HS in natural waters. The effects of HS on the mobilization dynamics of actinides are of particular interest in risk assessment of nuclear waste repositories. Here, we present conditional equilibrium binding constants (Kc, MHA) of thorium, hafnium, and zirconium-humic acid complexes from ligand competition experiments using capillary electrophoresis coupled with ICP-MS (CE- ICP-MS). Equilibrium dialysis ligand exchange (EDLE) experiments using size exclusion via a 1000 Damembrane were also performed to validate the CE-ICP-MS analysis. Experiments were performed at pH 3.5-7 with solutions containing one tetravalent metal (Th, Hf, or Zr), Elliot soil humic acid (EHA) or Pahokee peat humic acid (PHA), and EDTA. CE-ICP-MS and EDLE experiments yielded nearly identical binding constants for the metal- humic acid complexes, indicating that both methods are appropriate for examining metal speciation at conditions lower than neutral pH. We find that tetravalent metals form strong complexes with humic acids, with Kc, MHA several orders of magnitude above REE-humic complexes. Experiments were conducted at a range of dissolved HA concentrations to examine the effect of [HA]/[Th] molar ratio on Kc, MHA. At low metal loading conditions (i.e. elevated [HA]/[Th] ratios) the ThHA binding constant reached values that were not affected by the relative abundance of humic acid and thorium. The importance of [HA]/[Th] molar ratios on constraining the equilibrium of MHA complexation is apparent when our estimated Kc, MHA values attained at very low metal loading conditions are compared to existing literature data. Overall, experimental data suggest that the tetravalent transition metal/-actinide-humic acid complexation is important over a wide range of pH values, including mildly acidic conditions, and thus, these complexes should be included in speciation models.

Evidence for estrogen receptor beta-selective activity of Vitex agnus-castus and isolated flavones.

PubMed

Jarry, Hubertus; Spengler, Barbara; Porzel, Andrea; Schmidt, Juergen; Wuttke, Wolfgang; Christoffel, Volker

2003-10-01

Recent cell culture experiments indicated that extracts of Vitex agnus-castus (VAC) may contain yet unidentified phytoestrogens. Estrogenic actions are mediated via estrogen receptors (ER). To investigate whether VAC compounds bind to the currently known isoforms ERalpha or ERss, ligand binding assays (LBA) were performed. Subtype specific ER-LBA revealed a binding of VAC to ERss only. To isolate the ERss-selective compounds, the extract was fractionated by bio-guidance. The flavonoid apigenin was isolated and identified as the most active ERss-selective phytoestrogen in VAC. Other isolated compounds were vitexin and penduletin. These data demonstrate that the phytoestrogens in VAC are ERss-selective.

Spectral and computational features of the binding between riparins and human serum albumin

NASA Astrophysics Data System (ADS)

Camargo, Cintia Ramos; Caruso, Ícaro Putinhon; Gutierrez, Stanley Juan Chavez; Fossey, Marcelo Andres; Filho, José Maria Barbosa; Cornélio, Marinônio Lopes

2018-02-01

The green Brazilian bay leaf, a spice much prized in local cuisine (Aniba riparia, Lauraceae), contains chemical compounds presenting benzoyl-derivatives named riparins, which have anti-inflammatory, antimicrobial and anxiolytic properties. However, it is unclear what kind of interaction riparins perform with any molecular target. As a profitable target, human serum albumin (HSA) is one of the principal extracellular proteins, with an exceptional capacity to interact with several molecules, and it also plays a crucial role in the transport, distribution, and metabolism of a wide variety of endogenous and exogenous ligands. To outline the HSA-riparin interaction mechanism, spectroscopy and computational methods were synergistically applied. An evaluation through fluorescence spectroscopy showed that the emission, attributed to Trp 214, at 346 nm decreased with titrations of riparins. A static quenching mechanism was observed in the binding of riparins to HSA. Fluorescence experiments performed at 298, 308 and 318 K made it possible to conduct thermodynamic analysis indicating a spontaneous reaction in the complex formation (ΔG < 0). The enthalpy-entropy balance experiment with a molecular modeling calculation revealed that hydrophobic, hydrogen bond and non-specific interactions are present for riparin I-III with HSA. The set of results from fractional fluorescence changes obtained through Schatchard was inconclusive in establishing what kind of cooperativity is present in the interaction. To shed light upon the HSA-riparins complex, Hill's approach was utilized to distinguish the index of affinity and the binding constant. A correspondence between the molecular structures of riparins, due to the presence of the hydroxyl group in the B-ring, with thermodynamic parameters and index of affinity were observed. Riparin III performs an intramolecular hydrogen bond, which affects the Hill coefficient and the binding constant. Therefore, the presence of hydroxyl groups is capable of modulating the interaction between riparins and HSA. Site marker competitive experiments indicated Site I as being the most suitable, and the molecular modeling tools reinforced the experimental results detailing the participation of residues.

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

PubMed

Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

2018-02-01

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

When the Wedding March becomes sad: Semantic memory impairment for music in the semantic variant of primary progressive aphasia.

PubMed

Macoir, Joël; Berubé-Lalancette, Sarah; Wilson, Maximiliano A; Laforce, Robert; Hudon, Carol; Gravel, Pierre; Potvin, Olivier; Duchesne, Simon; Monetta, Laura

2016-12-01

Music can induce particular emotions and activate semantic knowledge. In the semantic variant of primary progressive aphasia (svPPA), semantic memory is impaired as a result of anterior temporal lobe (ATL) atrophy. Semantics is responsible for the encoding and retrieval of factual knowledge about music, including associative and emotional attributes. In the present study, we report the performance of two individuals with svPPA in three experiments. NG with bilateral ATL atrophy and ND with atrophy largely restricted to the left ATL. Experiment 1 assessed the recognition of musical excerpts and both patients were unimpaired. Experiment 2 studied the emotions conveyed by music and only NG showed impaired performance. Experiment 3 tested the association of semantic concepts to musical excerpts and both patients were impaired. These results suggest that the right ATL seems essential for the recognition of emotions conveyed by music and that the left ATL is involved in binding music to semantics. They are in line with the notion that the ATLs are devoted to the binding of different modality-specific properties and suggest that they are also differentially involved in the processing of factual and emotional knowledge associated with music.

Binding modes of environmental endocrine disruptors to human serum albumin: insights from STD-NMR, ITC, spectroscopic and molecular docking studies.

PubMed

Yang, Hongqin; Huang, Yanmei; Liu, Jiuyang; Tang, Peixiao; Sun, Qiaomei; Xiong, Xinnuo; Tang, Bin; He, Jiawei; Li, Hui

2017-09-11

Given that bisphenols have an endocrine-disrupting effect on human bodies, thoroughly exposing their potential effects at the molecular level is important. Saturation transfer difference (STD) NMR-based binding studies were performed to investigate the binding potential of two bisphenol representatives, namely, bisphenol B (BPB) and bisphenol E (BPE), toward human serum albumin (HSA). The relative STD (%) suggested that BPB and BPE show similar binding modes and orientations, in which the phenolic rings were spatially close to HSA binding site. ITC analysis results showed that BPB and BPE were bound to HSA with moderately strong binding affinity through electrostatic interactions and hydrogen bonds. The order of binding affinity of HSA for two test bisphenols is as follows: BPE > BPB. The results of fluorescence competitive experiments using 5-dimethylaminonaphthalene-1-sulfonamide and dansylsarcosine as competitors, combined with molecular docking indicated that both bisphenols are prone to attach to the binding site II in HSA. Spectroscopic results (FT-IR, CD, synchronous and 3D fluorescence spectra) showed that BPB/BPE induces different degrees of microenvironmental and conformational changes to HSA.

Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding.

PubMed

Goncharova, Iryna; Jašprová, Jana; Vítek, Libor; Urbanová, Marie

2015-12-01

The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp-HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment-HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA). Copyright © 2015 Elsevier Inc. All rights reserved.

A common perceptual temporal limit of binding synchronous inputs across different sensory attributes and modalities

PubMed Central

Fujisaki, Waka; Nishida, Shin'ya

2010-01-01

The human brain processes different aspects of the surrounding environment through multiple sensory modalities, and each modality can be subdivided into multiple attribute-specific channels. When the brain rebinds sensory content information (‘what’) across different channels, temporal coincidence (‘when’) along with spatial coincidence (‘where’) provides a critical clue. It however remains unknown whether neural mechanisms for binding synchronous attributes are specific to each attribute combination, or universal and central. In human psychophysical experiments, we examined how combinations of visual, auditory and tactile attributes affect the temporal frequency limit of synchrony-based binding. The results indicated that the upper limits of cross-attribute binding were lower than those of within-attribute binding, and surprisingly similar for any combination of visual, auditory and tactile attributes (2–3 Hz). They are unlikely to be the limits for judging synchrony, since the temporal limit of a cross-attribute synchrony judgement was higher and varied with the modality combination (4–9 Hz). These findings suggest that cross-attribute temporal binding is mediated by a slow central process that combines separately processed ‘what’ and ‘when’ properties of a single event. While the synchrony performance reflects temporal bottlenecks existing in ‘when’ processing, the binding performance reflects the central temporal limit of integrating ‘when’ and ‘what’ properties. PMID:20335212

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.

PubMed Central

Marciniak, R A; Garcia-Blanco, M A; Sharp, P A

1990-01-01

Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo. Images PMID:2333305

General odorant-binding proteins and sex pheromone guide larvae of Plutella xylostella to better food.

PubMed

Zhu, Jiao; Ban, Liping; Song, Li-Mei; Liu, Yang; Pelosi, Paolo; Wang, Guirong

2016-05-01

Olfaction of Lepidopteran larvae has received little attention, compared to the damage to crops done by insects at this stage. We report that larvae of the diamondback moth Plutella xylostella are attracted to their natural sex pheromone and to their major component (Z)-11-hexadecenal, but only in a food context. For such task they use two general odorant-binding proteins (GOBPs), abundantly expressed in the three major sensilla basiconica of the larval antenna, as shown by whole-mount immunostaining and immunocytochemistry experiments. None of the three genes encoding pheromone-binding proteins (PBPs) are expressed at this stage. Both recombinant GOBPs bind (Z)-11-hexadecenal and the corresponding alcohol, but not the acetate. Binding experiments performed with five mutants of GOBP2, where aromatic residues in the binding pocket were replaced with leucine showed that only one or two amino acid substitutions can completely abolish binding to the pheromone shifting the affinity to plant-derived compounds. We hypothesise that detection of their species-specific pheromone may direct larvae to the sites of foraging chosen by their mother when laying eggs, to find better food, as well as to reduce competition with individuals of the same or other species sharing the same host plant. We also provide evidence that GOBP2 is a narrowly tuned binding protein, whose affinity can be easily switched from linear pheromones to branched plants terpenoids, representing a tool better suited for the simple olfactory system of larvae, as compared to the more sophisticated organ of adults. Copyright © 2016 Elsevier Ltd. All rights reserved.

Object-based attention underlies the rehearsal of feature binding in visual working memory.

PubMed

Shen, Mowei; Huang, Xiang; Gao, Zaifeng

2015-04-01

Feature binding is a core concept in many research fields, including the study of working memory (WM). Over the past decade, it has been debated whether keeping the feature binding in visual WM consumes more visual attention than the constituent single features. Previous studies have only explored the contribution of domain-general attention or space-based attention in the binding process; no study so far has explored the role of object-based attention in retaining binding in visual WM. We hypothesized that object-based attention underlay the mechanism of rehearsing feature binding in visual WM. Therefore, during the maintenance phase of a visual WM task, we inserted a secondary mental rotation (Experiments 1-3), transparent motion (Experiment 4), or an object-based feature report task (Experiment 5) to consume the object-based attention available for binding. In line with the prediction of the object-based attention hypothesis, Experiments 1-5 revealed a more significant impairment for binding than for constituent single features. However, this selective binding impairment was not observed when inserting a space-based visual search task (Experiment 6). We conclude that object-based attention underlies the rehearsal of binding representation in visual WM. (c) 2015 APA, all rights reserved.

Role of NF-Kappa B Signaling in X-Box Binding Protein 1 (XBP1)-Mediated Antiestrogen Resistance in Breast Cancer

DTIC Science & Technology

2013-10-01

experiments, a statistically significant data is not yet available. Additional experiments are needed for us to be able to draw   conclu comb Figure...well-defined stress pathways, UPR and autophagy, are involved breast involution regulation. Using published gene expression array datasets from...performed involution time-course experiments using both low-dose drug interventions and an autophagy-related gene 7 (ATG7) deletion mouse model

Using attribute amnesia to test the limits of hyper-binding and associative deficits in working memory.

PubMed

McCormick-Huhn, John M; Chen, Hui; Wyble, Bradley P; Dennis, Nancy A

2018-02-01

Previous work has shown mixed evidence regarding age-related deficits for binding in working memory. The current study used the newly developed attribute amnesia effect (H. Chen & Wyble, 2015a) to test the associative-deficit hypothesis during working memory and to probe whether hyper-binding extends to include binding of de-selected information. In studies of attribute amnesia, participants use target attributes (e.g., identity, color) to demonstrate near ceiling levels of reporting of a second target attribute (e.g., location) across a series of trials (H. Chen & Wyble, 2015a, 2016). Yet, despite having just processed the target-defining attribute, they have difficulty reporting it on a surprise trial. This effect provides several predictions for associative binding in aging. The associative-deficit hypothesis predicts age-related decline on the surprise trial, whereas an extension of hyper-binding predicts age-related increase in performance in older adults. In Experiment 1, when working memory load was low, older adults demonstrated attribute amnesia equal to that found in younger adults. When load increased in Experiment 2, older adults again demonstrated attribute amnesia as well as an age deficit for reporting target attributes. In lieu of spontaneous binding, results suggest that expectancy plays a critical role in older adults' propensity to encode and bind target attributes in working memory. Results further suggest that expectancy alone is not enough for older adults to form bound representations when task demands are high. Taken together results revealed a boundary condition of hyper-binding and further provided conditional support for the associative-deficit hypothesis in working memory. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Ca(2+)-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay.

PubMed

Krasitskaya, V V; Korneeva, S I; Kudryavtsev, A N; Markova, S V; Stepanyuk, G A; Frank, L A

2011-11-01

The recombinant Ca(2+)-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.

Analytic second derivative of the energy for density-functional tight-binding combined with the fragment molecular orbital method

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

Nakata, Hiroya, E-mail: hiroya.nakata.gt@kyocera.jp; Nishimoto, Yoshio; Fedorov, Dmitri G.

2016-07-28

The analytic second derivative of the energy is developed for the fragment molecular orbital (FMO) method combined with density-functional tight-binding (DFTB), enabling simulations of infrared and Raman spectra of large molecular systems. The accuracy of the method is established in comparison to full DFTB without fragmentation for a set of representative systems. The performance of the FMO-DFTB Hessian is discussed for molecular systems containing up to 10 041 atoms. The method is applied to the study of the binding of α-cyclodextrin to polyethylene glycol, and the calculated IR spectrum of an epoxy amine oligomer reproduces experiment reasonably well.

A practical approach to automate randomized design of experiments for ligand-binding assays.

PubMed

Tsoi, Jennifer; Patel, Vimal; Shih, Judy

2014-03-01

Design of experiments (DOE) is utilized in optimizing ligand-binding assay by modeling factor effects. To reduce the analyst's workload and error inherent with DOE, we propose the integration of automated liquid handlers to perform the randomized designs. A randomized design created from statistical software was imported into custom macro converting the design into a liquid-handler worklist to automate reagent delivery. An optimized assay was transferred to a contract research organization resulting in a successful validation. We developed a practical solution for assay optimization by integrating DOE and automation to increase assay robustness and enable successful method transfer. The flexibility of this process allows it to be applied to a variety of assay designs.

Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

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

de Miranda Santos, I.K.; Pereira, M.E.

1984-09-01

Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various /sup 125/I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeusmore » and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.« less

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

PubMed Central

Tempelman, L A; Hammer, D A

1994-01-01

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear. Images FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 8 FIGURE 10 PMID:8038394

Effects of autoshaping procedures on 3H-8-OH-DPAT-labeled 5-HT1a binding and 125I-LSD-labeled 5-HT2a binding in rat brain.

PubMed

Tomie, Arthur; Di Poce, Jason; Aguado, Allison; Janes, Amy; Benjamin, Daniel; Pohorecky, Larissa

2003-06-13

Effects of experience with Pavlovian autoshaping procedures on lever-press autoshaping conditioned response (CR) performance and 3H-8-OH-DPAT-labeled binding of 5-HT(1a) receptors as well as 125I-LSD-labeled binding of 5-HT(2a) receptors were evaluated in four groups of male Long-Evans hooded rats. Two groups of rats (Group Paired High CR and Group Paired Low CR) received Pavlovian autoshaping procedures wherein the presentation of a lever (conditioned stimulus, CS) was followed by the response-independent presentation of food (unconditioned stimulus, US). Rats in Group Paired High CR (n=12) showed more rapid CR acquisition and higher asymptotic levels of lever-press autoshaping CR performance relative to rats in Group Low CR (n=12). Group Omission (n=9) received autoshaping with an omission contingency, such that performing the lever-press autoshaping CR resulted in the cancellation the food US, while Group Random (n=9) received presentations of lever CS and food US randomly with respect to one another. Though Groups Omission and Random did not differ in lever-press autoshaping CR performance, Group Omission showed significantly lower levels of 3H-8-OH-DPAT-labeled 5-HT(1a) binding in post-synaptic areas (frontal cortex, septum, caudate putamen), as well as significantly higher plasma corticosterone levels than Group Random. In addition, Group Random showed higher levels of 3H-8-OH-DPAT-labeled 5-HT(1a) binding in pre-synaptic somatodendritic autoreceptors on dorsal raphe nucleus relative to each of the other three groups. Autoradiographic analysis of 125I-LSD-labeled 5-HT(2a) receptor binding revealed no significant differences between Groups Paired High CR and Paired Low CR or between Groups Omission and Random in any brain regions.

Accounting for GC-content bias reduces systematic errors and batch effects in ChIP-seq data.

PubMed

Teng, Mingxiang; Irizarry, Rafael A

2017-11-01

The main application of ChIP-seq technology is the detection of genomic regions that bind to a protein of interest. A large part of functional genomics' public catalogs is based on ChIP-seq data. These catalogs rely on peak calling algorithms that infer protein-binding sites by detecting genomic regions associated with more mapped reads (coverage) than expected by chance, as a result of the experimental protocol's lack of perfect specificity. We find that GC-content bias accounts for substantial variability in the observed coverage for ChIP-seq experiments and that this variability leads to false-positive peak calls. More concerning is that the GC effect varies across experiments, with the effect strong enough to result in a substantial number of peaks called differently when different laboratories perform experiments on the same cell line. However, accounting for GC content bias in ChIP-seq is challenging because the binding sites of interest tend to be more common in high GC-content regions, which confounds real biological signals with unwanted variability. To account for this challenge, we introduce a statistical approach that accounts for GC effects on both nonspecific noise and signal induced by the binding site. The method can be used to account for this bias in binding quantification as well to improve existing peak calling algorithms. We use this approach to show a reduction in false-positive peaks as well as improved consistency across laboratories. © 2017 Teng and Irizarry; Published by Cold Spring Harbor Laboratory Press.

Perceptual learning shapes multisensory causal inference via two distinct mechanisms

PubMed Central

McGovern, David P.; Roudaia, Eugenie; Newell, Fiona N.; Roach, Neil W.

2016-01-01

To accurately represent the environment, our brains must integrate sensory signals from a common source while segregating those from independent sources. A reasonable strategy for performing this task is to restrict integration to cues that coincide in space and time. However, because multisensory signals are subject to differential transmission and processing delays, the brain must retain a degree of tolerance for temporal discrepancies. Recent research suggests that the width of this ‘temporal binding window’ can be reduced through perceptual learning, however, little is known about the mechanisms underlying these experience-dependent effects. Here, in separate experiments, we measure the temporal and spatial binding windows of human participants before and after training on an audiovisual temporal discrimination task. We show that training leads to two distinct effects on multisensory integration in the form of (i) a specific narrowing of the temporal binding window that does not transfer to spatial binding and (ii) a general reduction in the magnitude of crossmodal interactions across all spatiotemporal disparities. These effects arise naturally from a Bayesian model of causal inference in which learning improves the precision of audiovisual timing estimation, whilst concomitantly decreasing the prior expectation that stimuli emanate from a common source. PMID:27091411

Feature binding and attention in working memory: a resolution of previous contradictory findings.

PubMed

Allen, Richard J; Hitch, Graham J; Mate, Judit; Baddeley, Alan D

2012-01-01

We aimed to resolve an apparent contradiction between previous experiments from different laboratories, using dual-task methodology to compare effects of a concurrent executive load on immediate recognition memory for colours or shapes of items or their colour-shape combinations. Results of two experiments confirmed previous evidence that an irrelevant attentional load interferes equally with memory for features and memory for feature bindings. Detailed analyses suggested that previous contradictory evidence arose from limitations in the way recognition memory was measured. The present findings are inconsistent with an earlier suggestion that feature binding takes place within a multimodal episodic buffer Baddeley, ( 2000 ) and support a subsequent account in which binding takes place automatically prior to information entering the episodic buffer Baddeley, Allen, & Hitch, ( 2011 ). Methodologically, the results suggest that different measures of recognition memory performance (A', d', corrected recognition) give a converging picture of main effects, but are less consistent in detecting interactions. We suggest that this limitation on the reliability of measuring recognition should be taken into account in future research so as to avoid problems of replication that turn out to be more apparent than real.

Perceptual learning shapes multisensory causal inference via two distinct mechanisms.

PubMed

McGovern, David P; Roudaia, Eugenie; Newell, Fiona N; Roach, Neil W

2016-04-19

To accurately represent the environment, our brains must integrate sensory signals from a common source while segregating those from independent sources. A reasonable strategy for performing this task is to restrict integration to cues that coincide in space and time. However, because multisensory signals are subject to differential transmission and processing delays, the brain must retain a degree of tolerance for temporal discrepancies. Recent research suggests that the width of this 'temporal binding window' can be reduced through perceptual learning, however, little is known about the mechanisms underlying these experience-dependent effects. Here, in separate experiments, we measure the temporal and spatial binding windows of human participants before and after training on an audiovisual temporal discrimination task. We show that training leads to two distinct effects on multisensory integration in the form of (i) a specific narrowing of the temporal binding window that does not transfer to spatial binding and (ii) a general reduction in the magnitude of crossmodal interactions across all spatiotemporal disparities. These effects arise naturally from a Bayesian model of causal inference in which learning improves the precision of audiovisual timing estimation, whilst concomitantly decreasing the prior expectation that stimuli emanate from a common source.

Lack of effect of reserpine-induced dopamine depletion on the binding of the dopamine-D3 selective radioligand, [11C]RGH-1756.

PubMed

Sóvágó, Judit; Farde, Lars; Halldin, Christer; Schukin, Evgenij; Schou, Magnus; Laszlovszky, István; Kiss, Béla; Gulyás, Balázs

2005-10-15

The effect of reserpine induced dopamine depletion on the binding of the putative dopamine-D3 receptor ligand, [(11)C]RGH-1756 was examined in the monkey brain with positron emission tomography (PET). In a previous series of experiments, we have made an attempt to selectively label D3 receptors in the monkey brain using [(11)C]RGH-1756. Despite high selectivity and affinity of RGH-1756 in vitro, [(11)C]RGH-1756 displayed only low specific binding to D3 receptors in vivo. The aim of the present study was to examine whether low specific binding of [(11)C]RGH-1756 is caused by insufficient in vivo affinity of the ligand, or by high physiological occupancy of D3 receptors by endogenous dopamine (DA). PET experiments were performed in three monkeys under baseline conditions and after administration of reserpine (0.5 mg/kg). The results of the baseline measurements corresponded well to our earlier observations with [(11)C]RGH-1756. Reserpine caused no evident change in the regional distribution of [(11)C]RGH-1756 in the monkey brain, and no conspicuous regional accumulation of activity could be observed. After reserpine treatment there was no evident increase of specific binding and binding potential (BP) of [(11)C]RGH-1756. The lack of increased [(11)C]RGH-1756 binding after reserpine treatment indicates that competition with endogenous DA is not the predominant reason for the failure of the radioligand to label D3 receptors. Therefore, the low binding of [(11)C]RGH-1756 could largely be explained by the need for very high affinity of radioligand for D3 receptors in vivo, to obtain a suitable signal for the minute densities of D3 receptors expressed in the primate brain.

Expression of σ receptors of human urinary bladder tumor cells (RT-4 cells) and development of a competitive receptor binding assay for the determination of ligand affinity to human σ(2) receptors.

PubMed

Schepmann, Dirk; Lehmkuhl, Kirstin; Brune, Stefanie; Wünsch, Bernhard

2011-07-15

A selective competitive binding assay for the determination of the affinity of compounds to the human σ(2) receptor using 96-well multiplates and a solid state scintillator was developed. In the assay system, [(3)H]ditolylguanidine (DTG) was used as radioligand and membrane homogenates from human RT-4 cells physiologically expressing σ(2) receptors served as receptor material. In order to block the interaction of the unselective radioligand [(3)H]DTG with σ(1) receptors, all experiments were performed in the presence of the σ(1) selective ligand (+)-pentazocine. The density of σ(2) receptors of the cells was analyzed by a saturation experiment with [(3)H]DTG. The radioligand [(3)H]DTG was bound to a single, saturable site on human σ(2) receptors, resulting in a B(max) value of 2108±162fmol/mg protein and K(d)-value of 8.3±2.0nM. The expression of competing σ(1) receptors was evaluated by performing a saturation experiment using the σ(1) selective radioligand [(3)H](+)-pentazocine, which resulted in a B(max) value of 279±40fmol/mg protein and K(d) value of 13.4±1.6nM. For validation of the σ(2) binding assay, the K(i)-values of four σ(2) ligands (ditolylguanidine, haloperidol, rimczole and BMY-14802) were determined with RT-4 cell membrane preparations. The K(i) values obtained from these experiments are in good accordance with the K(i)-values obtained with rat liver membrane preparations as receptor material and with K(i) values given in the literature. Copyright © 2011 Elsevier B.V. All rights reserved.

Development and application of an algorithm to compute weighted multiple glycan alignments.

PubMed

Hosoda, Masae; Akune, Yukie; Aoki-Kinoshita, Kiyoko F

2017-05-01

A glycan consists of monosaccharides linked by glycosidic bonds, has branches and forms complex molecular structures. Databases have been developed to store large amounts of glycan-binding experiments, including glycan arrays with glycan-binding proteins. However, there are few bioinformatics techniques to analyze large amounts of data for glycans because there are few tools that can handle the complexity of glycan structures. Thus, we have developed the MCAW (Multiple Carbohydrate Alignment with Weights) tool that can align multiple glycan structures, to aid in the understanding of their function as binding recognition molecules. We have described in detail the first algorithm to perform multiple glycan alignments by modeling glycans as trees. To test our tool, we prepared several data sets, and as a result, we found that the glycan motif could be successfully aligned without any prior knowledge applied to the tool, and the known recognition binding sites of glycans could be aligned at a high rate amongst all our datasets tested. We thus claim that our tool is able to find meaningful glycan recognition and binding patterns using data obtained by glycan-binding experiments. The development and availability of an effective multiple glycan alignment tool opens possibilities for many other glycoinformatics analysis, making this work a big step towards furthering glycomics analysis. http://www.rings.t.soka.ac.jp. kkiyoko@soka.ac.jp. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities.

PubMed

Alqarni, Mohammed; Myint, Kyaw Zeyar; Tong, Qin; Yang, Peng; Bartlow, Patrick; Wang, Lirong; Feng, Rentian; Xie, Xiang-Qun

2014-09-26

We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor. Copyright © 2014 Elsevier Inc. All rights reserved.

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core RISC, composed of human Argonaute 2 and a guide RNA.

PubMed

Jo, Myung Hyun; Song, Ji-Joon; Hohng, Sungchul

2015-12-01

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection.

Mechanistic aspects of thioflavin-T self-aggregation and DNA binding: evidence for dimer attack on DNA grooves.

PubMed

Biancardi, A; Biver, T; Burgalassi, A; Mattonai, M; Secco, F; Venturini, M

2014-10-07

Thioflavin-T (TFT) is a fluorescent marker widely employed in biomedical research but the mechanism of its binding to polynucleotides has been poorly understood. This paper presents a study of the mechanisms of TFT self-aggregation and binding to DNA. Relaxation kinetics of TFT solutions show that the cyanine undergoes dimerization followed by dimer isomerisation. The interaction of TFT with DNA has been investigated using static methods, such as spectrophotometric and spectrofluorometric titrations under different conditions (salt content, temperature), fluorescence quenching, viscometric experiments and the T-jump relaxation method. The combined use of these techniques enabled us to show that the TFT monomer undergoes intercalation between the DNA base pairs and external binding according to a branched mechanism. Moreover, it has also been observed that, under dye excess conditions, the TFT dimer binds to the DNA grooves. The molecular structures of intercalated TFT and the groove-bound TFT dimer are obtained by performing QM/MM MD simulations.

Mapping and analysis of Caenorhabditis elegans transcription factor sequence specificities

PubMed Central

Narasimhan, Kamesh; Lambert, Samuel A; Yang, Ally WH; Riddell, Jeremy; Mnaimneh, Sanie; Zheng, Hong; Albu, Mihai; Najafabadi, Hamed S; Reece-Hoyes, John S; Fuxman Bass, Juan I; Walhout, Albertha JM; Weirauch, Matthew T; Hughes, Timothy R

2015-01-01

Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs. DOI: http://dx.doi.org/10.7554/eLife.06967.001 PMID:25905672

Ammonoxidised lignins as slow nitrogen-releasing soil amendments and CO₂-binding matrix.

PubMed

Liebner, Falk; Pour, Georg; de la Rosa Arranz, José Maria; Hilscher, André; Rosenau, Thomas; Knicker, Heike

2011-09-05

Nitrogen (N) is a major nutrient element controlling the cycling of organic matter in the biosphere. Its availability in soils is closely related to biological productivity. In order to reduce the negative environmental impact, associated with the application of mineral N-fertilizers, the use of ammonoxidised technical lignins is suggested. They can act as potential slow N-release fertilisers which concomitantly may increase C sequestration of soils by its potential to bind CO₂. The idea of our study was to combine an improved chemical characterisation of ammonoxidised ligneous matter as well as their CO₂-binding potential, with laboratory pot experiments, performed to enable an evaluation of their behaviour and stability during the biochemical reworking occurring in active soils.

CovalentDock Cloud: a web server for automated covalent docking.

PubMed

Ouyang, Xuchang; Zhou, Shuo; Ge, Zemei; Li, Runtao; Kwoh, Chee Keong

2013-07-01

Covalent binding is an important mechanism for many drugs to gain its function. We developed a computational algorithm to model this chemical event and extended it to a web server, the CovalentDock Cloud, to make it accessible directly online without any local installation and configuration. It provides a simple yet user-friendly web interface to perform covalent docking experiments and analysis online. The web server accepts the structures of both the ligand and the receptor uploaded by the user or retrieved from online databases with valid access id. It identifies the potential covalent binding patterns, carries out the covalent docking experiments and provides visualization of the result for user analysis. This web server is free and open to all users at http://docking.sce.ntu.edu.sg/.

Empirical entropic contributions in computational docking: evaluation in APS reductase complexes.

PubMed

Chang, Max W; Belew, Richard K; Carroll, Kate S; Olson, Arthur J; Goodsell, David S

2008-08-01

The results from reiterated docking experiments may be used to evaluate an empirical vibrational entropy of binding in ligand-protein complexes. We have tested several methods for evaluating the vibrational contribution to binding of 22 nucleotide analogues to the enzyme APS reductase. These include two cluster size methods that measure the probability of finding a particular conformation, a method that estimates the extent of the local energetic well by looking at the scatter of conformations within clustered results, and an RMSD-based method that uses the overall scatter and clustering of all conformations. We have also directly characterized the local energy landscape by randomly sampling around docked conformations. The simple cluster size method shows the best performance, improving the identification of correct conformations in multiple docking experiments. 2008 Wiley Periodicals, Inc.

CovalentDock Cloud: a web server for automated covalent docking

PubMed Central

Ouyang, Xuchang; Zhou, Shuo; Ge, Zemei; Li, Runtao; Kwoh, Chee Keong

2013-01-01

Covalent binding is an important mechanism for many drugs to gain its function. We developed a computational algorithm to model this chemical event and extended it to a web server, the CovalentDock Cloud, to make it accessible directly online without any local installation and configuration. It provides a simple yet user-friendly web interface to perform covalent docking experiments and analysis online. The web server accepts the structures of both the ligand and the receptor uploaded by the user or retrieved from online databases with valid access id. It identifies the potential covalent binding patterns, carries out the covalent docking experiments and provides visualization of the result for user analysis. This web server is free and open to all users at http://docking.sce.ntu.edu.sg/. PMID:23677616

Feature bindings are maintained in visual short-term memory without sustained focused attention.

PubMed

Delvenne, Jean-François; Cleeremans, Axel; Laloyaux, Cédric

2010-01-01

Does the maintenance of feature bindings in visual short-term memory (VSTM) require sustained focused attention? This issue was investigated in three experiments, in which memory for single features (i.e., colors or shapes) was compared with memory for feature bindings (i.e., the link between the color and shape of an object). Attention was manipulated during the memory retention interval with a retro-cue, which allows attention to be directed and focused on a subset of memory items. The retro-cue was presented 700 ms after the offset of the memory display and 700 ms before the onset of the test display. If the maintenance of feature bindings - but not of individual features - in memory requires sustained focused attention, the retro-cue should not affect memory performance. Contrary to this prediction, we found that both memory for feature bindings and memory for individual features were equally improved by the retro-cue. Therefore, this finding does not support the view that the sustained focused attention is needed to properly maintain feature bindings in VSTM.

Unexpected DNA affinity and sequence selectivity through core rigidity in guanidinium-based minor groove binders.

PubMed

Nagle, Padraic S; McKeever, Caitriona; Rodriguez, Fernando; Nguyen, Binh; Wilson, W David; Rozas, Isabel

2014-09-25

In this paper we report the design and biophysical evaluation of novel rigid-core symmetric and asymmetric dicationic DNA binders containing 9H-fluorene and 9,10-dihydroanthracene cores as well as the synthesis of one of these fluorene derivatives. First, the affinity toward particular DNA sequences of these compounds and flexible core derivatives was evaluated by means of surface plasmon resonance and thermal denaturation experiments finding that the position of the cations significantly influence the binding strength. Then their affinity and mode of binding were further studied by performing circular dichroism and UV studies and the results obtained were rationalized by means of DFT calculations. We found that the fluorene derivatives prepared have the ability to bind to the minor groove of certain DNA sequences and intercalate to others, whereas the dihydroanthracene compounds bind via intercalation to all the DNA sequences studied here.

Short term memory for single surface features and bindings in ageing: A replication study.

PubMed

Isella, Valeria; Molteni, Federica; Mapelli, Cristina; Ferrarese, Carlo

2015-06-01

In the present study we replicated a previous experiment investigating visuo-spatial short term memory binding in young and older healthy individuals, in the attempt to verify the pattern of impairment that can be observed in normal elderly for short term memory for single items vs short term memory for bindings. Assessing a larger sample size (25 young and 25 older subjects), using a more appropriate measure of accuracy for a change detection task (A'), and adding the evaluation of speed of performance, we confirmed that old normals show a decline in short term memory for bindings of shape and colour that is of comparable extent, and not major, to the decline in memory for single shapes and single colours. The absence of a specific deficit of short term memory for conjunctions of surface features seems to distinguish cognitive ageing from Alzheimer's Disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Fast and Efficient Fragment-Based Lead Generation by Fully Automated Processing and Analysis of Ligand-Observed NMR Binding Data.

PubMed

Peng, Chen; Frommlet, Alexandra; Perez, Manuel; Cobas, Carlos; Blechschmidt, Anke; Dominguez, Santiago; Lingel, Andreas

2016-04-14

NMR binding assays are routinely applied in hit finding and validation during early stages of drug discovery, particularly for fragment-based lead generation. To this end, compound libraries are screened by ligand-observed NMR experiments such as STD, T1ρ, and CPMG to identify molecules interacting with a target. The analysis of a high number of complex spectra is performed largely manually and therefore represents a limiting step in hit generation campaigns. Here we report a novel integrated computational procedure that processes and analyzes ligand-observed proton and fluorine NMR binding data in a fully automated fashion. A performance evaluation comparing automated and manual analysis results on (19)F- and (1)H-detected data sets shows that the program delivers robust, high-confidence hit lists in a fraction of the time needed for manual analysis and greatly facilitates visual inspection of the associated NMR spectra. These features enable considerably higher throughput, the assessment of larger libraries, and shorter turn-around times.

Docking glycosaminoglycans to proteins: analysis of solvent inclusion

NASA Astrophysics Data System (ADS)

Samsonov, Sergey A.; Teyra, Joan; Pisabarro, M. Teresa

2011-05-01

Glycosaminoglycans (GAGs) are anionic polysaccharides, which participate in key processes in the extracellular matrix by interactions with protein targets. Due to their charged nature, accurate consideration of electrostatic and water-mediated interactions is indispensable for understanding GAGs binding properties. However, solvent is often overlooked in molecular recognition studies. Here we analyze the abundance of solvent in GAG-protein interfaces and investigate the challenges of adding explicit solvent in GAG-protein docking experiments. We observe PDB GAG-protein interfaces being significantly more hydrated than protein-protein interfaces. Furthermore, by applying molecular dynamics approaches we estimate that about half of GAG-protein interactions are water-mediated. With a dataset of eleven GAG-protein complexes we analyze how solvent inclusion affects Autodock 3, eHiTs, MOE and FlexX docking. We develop an approach to de novo place explicit solvent into the binding site prior to docking, which uses the GRID program to predict positions of waters and to locate possible areas of solvent displacement upon ligand binding. To investigate how solvent placement affects docking performance, we compare these results with those obtained by taking into account information about the solvent position in the crystal structure. In general, we observe that inclusion of solvent improves the results obtained with these methods. Our data show that Autodock 3 performs best, though it experiences difficulties to quantitatively reproduce experimental data on specificity of heparin/heparan sulfate disaccharides binding to IL-8. Our work highlights the current challenges of introducing solvent in protein-GAGs recognition studies, which is crucial for exploiting the full potential of these molecules for rational engineering.

An integrative view of storage of low- and high-level visual dimensions in visual short-term memory.

PubMed

Magen, Hagit

2017-03-01

Efficient performance in an environment filled with complex objects is often achieved through the temporal maintenance of conjunctions of features from multiple dimensions. The most striking finding in the study of binding in visual short-term memory (VSTM) is equal memory performance for single features and for integrated multi-feature objects, a finding that has been central to several theories of VSTM. Nevertheless, research on binding in VSTM focused almost exclusively on low-level features, and little is known about how items from low- and high-level visual dimensions (e.g., colored manmade objects) are maintained simultaneously in VSTM. The present study tested memory for combinations of low-level features and high-level representations. In agreement with previous findings, Experiments 1 and 2 showed decrements in memory performance when non-integrated low- and high-level stimuli were maintained simultaneously compared to maintaining each dimension in isolation. However, contrary to previous findings the results of Experiments 3 and 4 showed decrements in memory performance even when integrated objects of low- and high-level stimuli were maintained in memory, compared to maintaining single-dimension objects. Overall, the results demonstrate that low- and high-level visual dimensions compete for the same limited memory capacity, and offer a more comprehensive view of VSTM.

A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects E scherichia coli from bile salt stress

PubMed Central

Paul, Stephanie; Alegre, Kamela O; Holdsworth, Scarlett R; Rice, Matthew; Brown, James A; McVeigh, Paul; Kelly, Sharon M; Law, Christopher J

2014-01-01

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as E scherichia coli. Although the tripartite AcrAB–TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E . coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E . coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB–TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H+ antiport. PMID:24684269

Face Memory: Implications for theories of binding items to context

PubMed Central

Reder, L. M.; Victoria, L. W.; Manelis, A.; Oates, J. M.; Dutcher, J. M.; Bates, J. T.; Cook, S.; Aizenstein, H. A.; Quinlan, J.; Gyulai, F.

2014-01-01

Two experiments tested the hypothesis that it is easier to bind a stimulus to context when the stimulus already has a stable (i.e., pre-existing) memory representation by comparing episodic memory of faces of celebrities vs. unknown individuals. Each face was superimposed on a picture of a well-known location (e.g., Eiffel Tower) during encoding and at a later unexpected recognition test but the background could change from encoding to test. Although recognition was to be based on the face, irrespective of background, performance was better when encoding context was reinstated. Further, a given background could be shown with many faces ("high fan") or only a few ("low fan") and this variable modulated the value added of context reinstatement. Importantly, manipulations of context only mattered for famous faces. As predicted, these effects were observed in recollection ("Remember") responses not in familiarity (“Know”) responses. Experiment 2 used the same design except that half of the subjects were administered midazolam, a drug that produces temporary anterograde amnesia, prior to encoding faces and backgrounds. Subjects injected with saline (control condition) showed the same pattern as Experiment 1; however subjects injected with midazolam showed a large decrease in the use of the "Remember" responses for famous faces and neither context reinstatement nor background fan affected performance. These results support the view that it is easier to bind stimuli to context when stimuli have a pre-existing, stable memory representation (e.g., faces of people whose identity we know) than when stimuli do not have pre-existing, stable memory representations. PMID:23395827

Optimization of Polyplex Formation between DNA Oligonucleotide and Poly(ʟ-Lysine): Experimental Study and Modeling Approach.

PubMed

Vasiliu, Tudor; Cojocaru, Corneliu; Rotaru, Alexandru; Pricope, Gabriela; Pinteala, Mariana; Clima, Lilia

2017-06-17

The polyplexes formed by nucleic acids and polycations have received a great attention owing to their potential application in gene therapy. In our study, we report experimental results and modeling outcomes regarding the optimization of polyplex formation between the double-stranded DNA (dsDNA) and poly(ʟ-Lysine) (PLL). The quantification of the binding efficiency during polyplex formation was performed by processing of the images captured from the gel electrophoresis assays. The design of experiments (DoE) and response surface methodology (RSM) were employed to investigate the coupling effect of key factors (pH and N/P ratio) affecting the binding efficiency. According to the experimental observations and response surface analysis, the N/P ratio showed a major influence on binding efficiency compared to pH. Model-based optimization calculations along with the experimental confirmation runs unveiled the maximal binding efficiency (99.4%) achieved at pH 5.4 and N/P ratio 125. To support the experimental data and reveal insights of molecular mechanism responsible for the polyplex formation between dsDNA and PLL, molecular dynamics simulations were performed at pH 5.4 and 7.4.

Optimization of Polyplex Formation between DNA Oligonucleotide and Poly(l-Lysine): Experimental Study and Modeling Approach

PubMed Central

Vasiliu, Tudor; Cojocaru, Corneliu; Rotaru, Alexandru; Pricope, Gabriela; Pinteala, Mariana; Clima, Lilia

2017-01-01

The polyplexes formed by nucleic acids and polycations have received a great attention owing to their potential application in gene therapy. In our study, we report experimental results and modeling outcomes regarding the optimization of polyplex formation between the double-stranded DNA (dsDNA) and poly(l-Lysine) (PLL). The quantification of the binding efficiency during polyplex formation was performed by processing of the images captured from the gel electrophoresis assays. The design of experiments (DoE) and response surface methodology (RSM) were employed to investigate the coupling effect of key factors (pH and N/P ratio) affecting the binding efficiency. According to the experimental observations and response surface analysis, the N/P ratio showed a major influence on binding efficiency compared to pH. Model-based optimization calculations along with the experimental confirmation runs unveiled the maximal binding efficiency (99.4%) achieved at pH 5.4 and N/P ratio 125. To support the experimental data and reveal insights of molecular mechanism responsible for the polyplex formation between dsDNA and PLL, molecular dynamics simulations were performed at pH 5.4 and 7.4. PMID:28629130

Thermodynamic Exploration of Eosin-Lysozyme Binding: A Physical Chemistry and Biochemistry Laboratory Experiment

ERIC Educational Resources Information Center

Huisman, Andrew J.; Hartsell, Lydia R.; Krueger, Brent P.; Pikaart, Michael J.

2010-01-01

We developed a modular pair of experiments for use in the undergraduate physical chemistry and biochemistry laboratories. Both experiments examine the thermodynamics of the binding of a small molecule, eosin Y, to the protein lysozyme. The assay for binding is the quenching of lysozyme fluorescence by eosin through resonant energy transfer. In…

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

PubMed

Schöne, Tobias; Grimm, Lena Lisbeth; Sakai, Naoki; Zhang, Linlin; Hilgenfeld, Rolf; Peters, Thomas

2017-10-01

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3 pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3 pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3 pro , we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3 pro . Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC 50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3 pro . To identify the matching protein binding site, chemical shift perturbation studies employing 1 H, 15 N-TROSY-HSQC experiments with uniformly 2 H, 15 N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3 pro inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study

PubMed Central

2014-01-01

Endotoxins are part of the cell wall of Gram-negative bacteria. They are potent immune stimulators and can lead to death if present in high concentrations. Feed additives, which bind endotoxins in the gastrointestinal tract of animals, could help to prevent their negative impact. The objective of our study was to determine the potential of a bentonite (Bentonite 1), a sodium bentonite (Bentonite 2), a chemically treated smectite (Organoclay 1) and a modified attapulgite (Organoclay 2) to bind endotoxins in vitro. Polymyxin B served as positive control. The kinetic chromogenic Limulus Amebocyte lysate test was adapted to measure endotoxin activity. Firstly, a single sorption experiment (10 endotoxin units/mL (EU/mL)) was performed. Polymyxin B and organoclays showed 100% binding efficiency. Secondly, the adsorption efficiency of sorbents in aqueous solution with increasing endotoxin concentrations (2,450 – 51,700 EU/mL) was investigated. Organoclay 1 (0.1%) showed a good binding efficiency in aqueous solution (average 81%), whereas Bentonite 1 (0.1%) obtained a lower binding efficiency (21-54%). The following absorbent capacities were calculated in highest endotoxin concentration: 5.59 mg/g (Organoclay 1) > 3.97 mg/g (Polymyxin B) > 2.58mg/g (Organoclay 2) > 1.55 mg/g (Bentonite 1) > 1.23 mg/g (Bentonite 2). Thirdly, a sorption experiment in artificial intestinal fluid was conducted. Especially for organoclays, which are known to be unspecific adsorbents, the endotoxin binding capacity was significantly reduced. In contrast, Bentonite 1 showed comparable results in artificial intestinal fluid and aqueous solution. Based on the results of this in vitro study, the effect of promising clay minerals will be investigated in in vivo trials. PMID:24383578

Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels.

PubMed

Kinde, Monica N; Bu, Weiming; Chen, Qiang; Xu, Yan; Eckenhoff, Roderic G; Tang, Pei

2016-03-01

Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1β3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.

Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

PubMed Central

Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

2016-01-01

The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

PubMed

Banasik, Michał; Sachadyn, Paweł

2016-09-01

A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

(15)N and (13)C group-selective techniques extend the scope of STD NMR detection of weak host-guest interactions and ligand screening.

PubMed

Kövér, Katalin E; Wéber, Edit; Martinek, Tamás A; Monostori, Eva; Batta, Gyula

2010-10-18

Saturation transfer difference (STD) is a valuable tool for studying the binding of small molecules to large biomolecules and for obtaining detailed information on the binding epitopes. Here, we demonstrate that the proposed (15)N/(13)C variants of group-selective, "GS-STD" experiments provide a powerful approach to mapping the binding epitope of a ligand even in the absence of efficient spin diffusion within the target protein. Therefore, these experimental variants broaden the scope of STD studies to smaller and/or more-dynamic targets. The STD spectra obtained in four different experimental setups (selective (1)H STD, (15)N GS-STD, (13)C(Ar) and (13)C(aliphatic) GS-STD approaches) revealed that the signal-intensity pattern of the difference spectra is affected by both the type and the spatial distribution of the excited "transmitter" atoms, as well as by the efficiency of the spin-diffusion-mediated magnetization transfer. The performance of the experiments is demonstrated on a system by using the lectin, galectin-1 and its carbohydrate ligand, lactose.

Trace Metal-Humic Complexes in Natural Waters: Insights From Speciation Experiments

NASA Astrophysics Data System (ADS)

Stern, J. C.; Salters, V.; Sonke, J.

2006-12-01

The DOM cycle is intimately linked to the cycling and bioavailability of trace metals in aqueous environments. The presence or absence of DOM in the water column can determined whether trace elements will be present in limited quantities as a nutrient, or in surplus quantities as a toxicant. Humic substances (HS), which represent the refractory products of DOM degradation, strongly affect the speciation of trace metals in natural waters. To simulate metal-HS interactions in nature, experiments must be carried out using trace metal concentrations. Sensitive detection systems such as ICP-MS make working with small (nanomolar) concentrations possible. Capillary electrophoresis coupled with ICP-MS (CE-ICP-MS) has recently been identified as a rapid and accurate method to separate metal species and calculate conditional binding constants (log K_c) of metal-humic complexes. CE-ICP-MS was used to measure partitioning of metals between humic substances and a competing ligand (EDTA) and calculate binding constants of rare earth element (REE) and Th, Hf, and Zr-humic complexes at pH 3.5-8 and ionic strength of 0.1. Equilibrium dialysis ligand exchange (EDLE) experiments to validate the CE-ICP-MS method were performed to separate the metal-HS and metal-EDTA species by partitioning due to size exclusion via diffusion through a 1000 Da membrane. CE-ICP-MS experiments were also conducted to compare binding constants of REE with humic substances of various origin, including soil, peat, and aquatic DOM. Results of our experiments show an increase in log K_c with decrease in ionic radius for REE-humic complexes (the lanthanide contraction effect). Conditional binding constants of tetravalent metal-humic complexes were found to be several orders of magnitude higher than REE-humic complexes, indicating that tetravalent metals have a very strong affinity for humic substances. Because thorium is often used as a proxy for the tetravalent actinides, Th-HS binding constants can allow us to assess the importance of tetravalent actinide-humic complexes in groundwater transport from nuclear repositories. Our results suggest that tetravalent actinide-humic complexes couild be more important to account for in predictive speciation models than previously thought.

Surface Passivation in Empirical Tight Binding

NASA Astrophysics Data System (ADS)

He, Yu; Tan, Yaohua; Jiang, Zhengping; Povolotskyi, Michael; Klimeck, Gerhard; Kubis, Tillmann

2016-03-01

Empirical Tight Binding (TB) methods are widely used in atomistic device simulations. Existing TB methods to passivate dangling bonds fall into two categories: 1) Method that explicitly includes passivation atoms is limited to passivation with atoms and small molecules only. 2) Method that implicitly incorporates passivation does not distinguish passivation atom types. This work introduces an implicit passivation method that is applicable to any passivation scenario with appropriate parameters. This method is applied to a Si quantum well and a Si ultra-thin body transistor oxidized with SiO2 in several oxidation configurations. Comparison with ab-initio results and experiments verifies the presented method. Oxidation configurations that severely hamper the transistor performance are identified. It is also shown that the commonly used implicit H atom passivation overestimates the transistor performance.

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Rezaei Darestani, Reza; Winter, Philip; Kitova, Elena N.; Tuszynski, Jack A.; Klassen, John S.

2016-05-01

Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

Single molecule junction conductance and binding geometry

NASA Astrophysics Data System (ADS)

Kamenetska, Maria

This Thesis addresses the fundamental problem of controlling transport through a metal-organic interface by studying electronic and mechanical properties of single organic molecule-metal junctions. Using a Scanning Tunneling Microscope (STM) we image, probe energy-level alignment and perform STM-based break junction (BJ) measurements on molecules bound to a gold surface. Using Scanning Tunneling Microscope-based break-junction (STM-BJ) techniques, we explore the effect of binding geometry on single-molecule conductance by varying the structure of the molecules, metal-molecule binding chemistry and by applying sub-nanometer manipulation control to the junction. These experiments are performed both in ambient conditions and in ultra high vacuum (UHV) at cryogenic temperatures. First, using STM imaging and scanning tunneling spectroscopy (STS) measurements we explore binding configurations and electronic properties of an amine-terminated benzene derivative on gold. We find that details of metal-molecule binding affect energy-level alignment at the interface. Next, using the STM-BJ technique, we form and rupture metal-molecule-metal junctions ˜104 times to obtain conductance-vs-extension curves and extract most likely conductance values for each molecule. With these measurements, we demonstrated that the control of junction conductance is possible through a choice of metal-molecule binding chemistry and sub-nanometer positioning. First, we show that molecules terminated with amines, sulfides and phosphines bind selectively on gold and therefore demonstrate constant conductance levels even as the junction is elongated and the metal-molecule attachment point is modified. Such well-defined conductance is also obtained with paracyclophane molecules which bind to gold directly through the pi system. Next, we are able to create metal-molecule-metal junctions with more than one reproducible conductance signatures that can be accessed by changing junction geometry. In the case of pyridine-linked molecules, conductance can be reliably switched between two distinct conductance states using sub-nanometer mechanical manipulation. Using a methyl sulfide linker attached to an oligoene backbone, we are able to create a 3-nm-long molecular potentiometer, whose resistance can be tuned exponentially with Angstom-scale modulations in metal-molecule configuration. These experiments points to a new paradigm for attaining reproducible electrical characteristics of metal-organic devices which involves controlling linker-metal chemistry rather than fabricating identically structured metal-molecule interfaces. By choosing a linker group which is either insensitive to or responds reproducibly to changes in metal-molecule configuration, one can design single molecule devices with functionality more complex than a simple resistor. These ambient temperature experiments were combined with UHV conductance measurements performed in a commercial STM on amine-terminated benzene derivatives which conduct through a non-resonant tunneling mechanism, at temperatures varying from 5 to 300 Kelvin. Our results indicate that while amine-gold binding remains selective irrespective of environment, conductance is not temperature independent, in contrast to what is expected for a tunneling mechanism. Furthermore, using temperature-dependent measurements in ambient conditions we find that HOMO-conducting amines and LUMO-conducting pyridines show opposite dependence of conductance on temperature. These results indicate that energy-level alignment between the molecule and the electrodes changes as a result of varying electrode structure at different temperatures. We find that temperature can serve as a knob with which to tune transport properties of single molecule-metal junctions.

Feature binding in visual short-term memory is unaffected by task-irrelevant changes of location, shape, and color.

PubMed

Logie, Robert H; Brockmole, James R; Jaswal, Snehlata

2011-01-01

Three experiments used a change detection paradigm across a range of study-test intervals to address the respective contributions of location, shape, and color to the formation of bindings of features in sensory memory and visual short-term memory (VSTM). In Experiment 1, location was designated task irrelevant and was randomized between study and test displays. The task was to detect changes in the bindings between shape and color. In Experiments 2 and 3, shape and color, respectively, were task irrelevant and randomized, with bindings tested between location and color (Experiment 2) and location and shape (Experiment 3). At shorter study-test intervals, randomizing location was most disruptive, followed by shape and then color. At longer intervals, randomizing any task-irrelevant feature had no impact on change detection for bindings between features, and location had no special role. Results suggest that location is crucial for initial perceptual binding but loses that special status once representations are formed in VSTM, which operates according to different principles, than do visual attention and perception.

Introducing a simple model system for binding studies of known and novel inhibitors of AMPK: a therapeutic target for prostate cancer.

PubMed

Kumar, Rakesh; Maurya, Ranjana; Saran, Shweta

2018-02-23

Prostate cancer (PC) is one of the leading cancers in men, raising a serious health issue worldwide. Due to lack of suitable biomarker, their inhibitors and the platform for testing those inhibitors result in poor prognosis of PC. AMP-activated protein kinase (AMPK) is a highly conserved protein kinase found in eukaryotes that is involved in growth and development, and also acts as a therapeutic target for PC. The aim of the present study is to identify novel potent inhibitors of AMPK and propose a simple cellular model system for understanding its biology. Structural modelling and MD simulations were performed to construct and refine the 3D models of Dictyostelium and human AMPK. Binding mechanisms of different drug compounds were studied by performing molecular docking, molecular dynamics and MM-PBSA methods. Two novel drugs were isolated having higher binding affinity over the known drugs and hydrophobic forces that played a key role during protein-ligand interactions. The study also explored the simple cellular model system for drug screening and understanding the biology of a therapeutic target by performing in vitro experiments.

Impact of domain knowledge on blinded predictions of binding energies by alchemical free energy calculations

NASA Astrophysics Data System (ADS)

Mey, Antonia S. J. S.; Jiménez, Jordi Juárez; Michel, Julien

2018-01-01

The Drug Design Data Resource (D3R) consortium organises blinded challenges to address the latest advances in computational methods for ligand pose prediction, affinity ranking, and free energy calculations. Within the context of the second D3R Grand Challenge several blinded binding free energies predictions were made for two congeneric series of Farsenoid X Receptor (FXR) inhibitors with a semi-automated alchemical free energy calculation workflow featuring FESetup and SOMD software tools. Reasonable performance was observed in retrospective analyses of literature datasets. Nevertheless, blinded predictions on the full D3R datasets were poor due to difficulties encountered with the ranking of compounds that vary in their net-charge. Performance increased for predictions that were restricted to subsets of compounds carrying the same net-charge. Disclosure of X-ray crystallography derived binding modes maintained or improved the correlation with experiment in a subsequent rounds of predictions. The best performing protocols on D3R set1 and set2 were comparable or superior to predictions made on the basis of analysis of literature structure activity relationships (SAR)s only, and comparable or slightly inferior, to the best submissions from other groups.

footprintDB: a database of transcription factors with annotated cis elements and binding interfaces.

PubMed

Sebastian, Alvaro; Contreras-Moreira, Bruno

2014-01-15

Traditional and high-throughput techniques for determining transcription factor (TF) binding specificities are generating large volumes of data of uneven quality, which are scattered across individual databases. FootprintDB integrates some of the most comprehensive freely available libraries of curated DNA binding sites and systematically annotates the binding interfaces of the corresponding TFs. The first release contains 2422 unique TF sequences, 10 112 DNA binding sites and 3662 DNA motifs. A survey of the included data sources, organisms and TF families was performed together with proprietary database TRANSFAC, finding that footprintDB has a similar coverage of multicellular organisms, while also containing bacterial regulatory data. A search engine has been designed that drives the prediction of DNA motifs for input TFs, or conversely of TF sequences that might recognize input regulatory sequences, by comparison with database entries. Such predictions can also be extended to a single proteome chosen by the user, and results are ranked in terms of interface similarity. Benchmark experiments with bacterial, plant and human data were performed to measure the predictive power of footprintDB searches, which were able to correctly recover 10, 55 and 90% of the tested sequences, respectively. Correctly predicted TFs had a higher interface similarity than the average, confirming its diagnostic value. Web site implemented in PHP,Perl, MySQL and Apache. Freely available from http://floresta.eead.csic.es/footprintdb.

Student Assessment and Grade Retention: Evidence from a Natural Experiment

ERIC Educational Resources Information Center

Kornfeld, Marlies; Ochsen, Carsten

2017-01-01

In several countries, students are tracked into secondary school types. This paper studies whether parents or teachers assess students' potential performance more adequately. We evaluate a reform in the German state of North Rhine-Westphalia in 2006. The reform replaced parents' choice about their children's school type by a binding teacher…

Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase.

PubMed

Volkán-Kacsó, Sándor; Marcus, Rudolph A

2016-10-25

A recently proposed chemomechanical group transfer theory of rotary biomolecular motors is applied to treat single-molecule controlled rotation experiments. In these experiments, single-molecule fluorescence is used to measure the binding and release rate constants of nucleotides by monitoring the occupancy of binding sites. It is shown how missed events of nucleotide binding and release in these experiments can be corrected using theory, with F 1 -ATP synthase as an example. The missed events are significant when the reverse rate is very fast. Using the theory the actual rate constants in the controlled rotation experiments and the corrections are predicted from independent data, including other single-molecule rotation and ensemble biochemical experiments. The effective torsional elastic constant is found to depend on the binding/releasing nucleotide, and it is smaller for ADP than for ATP. There is a good agreement, with no adjustable parameters, between the theoretical and experimental results of controlled rotation experiments and stalling experiments, for the range of angles where the data overlap. This agreement is perhaps all the more surprising because it occurs even though the binding and release of fluorescent nucleotides is monitored at single-site occupancy concentrations, whereas the stalling and free rotation experiments have multiple-site occupancy.

Is It Reliable to Take the Molecular Docking Top Scoring Position as the Best Solution without Considering Available Structural Data?

PubMed

Ramírez, David; Caballero, Julio

2018-04-28

Molecular docking is the most frequently used computational method for studying the interactions between organic molecules and biological macromolecules. In this context, docking allows predicting the preferred pose of a ligand inside a receptor binding site. However, the selection of the “best” solution is not a trivial task, despite the widely accepted selection criterion that the best pose corresponds to the best energy score. Here, several rigid-target docking methods were evaluated on the same dataset with respect to their ability to reproduce crystallographic binding orientations, to test if the best energy score is a reliable criterion for selecting the best solution. For this, two experiments were performed: (A) to reconstruct the ligand-receptor complex by performing docking of the ligand in its own crystal structure receptor (defined as self-docking), and (B) to reconstruct the ligand-receptor complex by performing docking of the ligand in a crystal structure receptor that contains other ligand (defined as cross-docking). Root-mean square deviation (RMSD) was used to evaluate how different the obtained docking orientation is from the corresponding co-crystallized pose of the same ligand molecule. We found that docking score function is capable of predicting crystallographic binding orientations, but the best ranked solution according to the docking energy is not always the pose that reproduces the experimental binding orientation. This happened when self-docking was achieved, but it was critical in cross-docking. Taking into account that docking is typically used with predictive purposes, during cross-docking experiments, our results indicate that the best energy score is not a reliable criterion to select the best solution in common docking applications. It is strongly recommended to choose the best docking solution according to the scoring function along with additional structural criteria described for analogue ligands to assure the selection of a correct docking solution.

Binding and degradation of 125I-gastrin by plasma membranes from homogenized rat gastric mucosa.

PubMed

Kleveland, P M; Waldum, H L

1986-06-01

Binding of 125I-gastrin to the 270-30,000 g fraction from homogenized rat oxyntic mucosa was studied. 'Specific' binding was calculated by subtracting the binding at excess cold gastrin from the binding with labelled gastrin (250 pM) only. At 30 degrees C specific binding rose rapidly to a short-lived maximum before falling gradually, whereas at 15 degrees C and 0 degree C specific binding rose gradually to a higher plateau level. The reduced binding at 30 degrees C could be caused by degradation of either the tracer or the binding site or by a combination of these two events. Degradation of 125I-gastrin was evaluated by trichloroacetic acid (TCA) precipitation, fast protein liquid chromatography, and binding to a gastrin antibody (immunoreactivity). The effect of incubation on the binding site was evaluated by preincubation of the homogenate fraction before adding gastrin. In separate studies, the proteolytic activity of the homogenate fraction was studied by TCA precipitation of radioactive casein. Different enzyme inhibitors tested were virtually ineffective in preventing gastrin and casein degradation. Only lowering the incubation temperature to 15 degrees C or lower could prevent this degradation. The reduced and transient binding of 125I-gastrin at 30 degrees C most probably reflects tracer degradation. Accordingly, the gastrin binding experiments were performed at 15 degrees C. Only homogenates from the oxyntic area of the stomach bound 125I-gastrin specifically and with a Kd of 0.8 nM (Scatchard analysis). However, micromolar concentrations of unlabelled gastrin were required to inhibit half maximal binding of the tracer. The tracer binding was unaffected by secretin, slightly reduced by a CCK-9 analogue, and more markedly reduced by pentagastrin.

An in situ method to quantitatively determine dissolved free drug concentrations in vitro in the presence of polymer excipients using pulsatile microdialysis (PMD).

PubMed

Vejani, Charchil; Bellantone, Robert A

2015-12-30

In drug formulations containing polymer excipients, the effects of the polymer on the dissolved free drug concentration and resulting dissolution or release can be important, especially for poorly soluble drugs. In this study, an in vitro method based on pulsatile microdialysis (PMD) was developed to quantitatively determine dissolved free concentrations of drugs in the presence of polymers in aqueous media in situ (e.g., in place within the system being characterized). Formulations were made by dissolving various ratios of the drug griseofulvin and polymer PVP K30 in water and allowing the mix to equilibrate. A PMD probe was immersed in each mixture and the dissolved free drug concentrations were determined in the PMD samples. The experimental procedure and the equations used for data analysis are presented. To assess the consistency of data, a binding model was fit to the data obtained using PMD by calculating the dissolved free drug fraction fD for each drug-polymer ratio in solution, and obtaining the product of the binding stoichiometry and binding constant (νK per mole of polymer) from the slope of a plot of (1-fD)/fD vs. the molar polymer concentration. For comparison, equilibrium binding experiments were also performed at 23C, and the determined value of νK was similar to the value found using PMD. Experiments were performed at three temperatures, and a plot of ln (νK) vs. 1/T was linear and a binding enthalpy of -110.9±4.4J/mol of monomer was calculated from its slope. It was concluded that PMD can be used to determine the dissolved free drug concentrations in situ, which allows characterization of the drug-polymer interaction, even for low drug concentrations. This information may be important in modeling the dissolution or release of drugs from formulations containing polymers. Copyright © 2015 Elsevier B.V. All rights reserved.

How much does emotional valence of action outcomes affect temporal binding?

PubMed

Moreton, Joshua; Callan, Mitchell J; Hughes, Gethin

2017-03-01

Temporal binding refers to the compression of the perceived time interval between voluntary actions and their sensory consequences. Research suggests that the emotional content of an action outcome can modulate the effects of temporal binding. We attempted to conceptually replicate these findings using a time interval estimation task and different emotionally-valenced action outcomes (Experiments 1 and 2) than used in previous research. Contrary to previous findings, we found no evidence that temporal binding was affected by the emotional valence of action outcomes. After validating our stimuli for equivalence of perceived emotional valence and arousal (Experiment 3), in Experiment 4 we directly replicated Yoshie and Haggard's (2013) original experiment using sound vocalizations as action outcomes and failed to detect a significant effect of emotion on temporal binding. These studies suggest that the emotional valence of action outcomes exerts little influence on temporal binding. The potential implications of these findings are discussed. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Red Blood Cell Hematocrit Influences Platelet Adhesion Rate in a Microchannel

NASA Astrophysics Data System (ADS)

Spann, Andrew; Campbell, James; Fitzgibbon, Sean; Rodriguez, Armando; Shaqfeh, Eric

2014-11-01

The creation of a blood clot to stop bleeding involves platelets forming a plug at the site of injury. Red blood cells indirectly play a role in ensuring that the distribution of platelets across the height of the channel is not uniform - the contrast in deformability and size between platelets and red blood cells allows the platelets to preferentially marginate close to the walls. We perform 3D boundary integral simulations of a suspension of platelets and red blood cells in a periodic channel with a model that allows for platelet binding at the walls. The relative rate of platelet activity with varying hematocrit (volume fraction of red blood cells) is compared to experiments in which red blood cells and platelets flow through a channel coated with von Willebrand factor. In the simulations as well as the experiments, a decrease in hematocrit of red blood cells is found to reduce the rate at which platelets adhere to the channel wall in a manner that is both qualitatively and quantitatively similar. We conclude with a discussion of the tumbling and wobbling motions of platelets in 3D leading up to the time at which the platelets bind to the wall. Funded by Stanford Army High Performance Computing Research Center, experiments by US Army Institute of Surgical Research.

Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets.

PubMed

Bachelet, Laure; Bertholon, Isabelle; Lavigne, Damien; Vassy, Roger; Jandrot-Perrus, Martine; Chaubet, Frédéric; Letourneur, Didier

2009-02-01

P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate. Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators. The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC(50) of 20 nM as compared to 400 nM for heparin and <25000 nM for dextran sulfate. It exhibited the highest affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the K(D) of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction. Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains♦

PubMed Central

Tanaka, Hiroaki; Akagi, Ken-ichi; Oneyama, Chitose; Tanaka, Masakazu; Sasaki, Yuichi; Kanou, Takashi; Lee, Young-Ho; Yokogawa, Daisuke; Dobenecker, Marc-Werner; Nakagawa, Atsushi; Okada, Masato; Ikegami, Takahisa

2013-01-01

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain. PMID:23548896

Pseudomonas aeruginosa AmrZ Binds to Four Sites in the algD Promoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation.

PubMed

Xu, Binjie; Soukup, Randal J; Jones, Christopher J; Fishel, Richard; Wozniak, Daniel J

2016-10-01

During late stages of cystic fibrosis pulmonary infections, Pseudomonas aeruginosa often overproduces the exopolysaccharide alginate, protecting the bacterial community from host immunity and antimicrobials. The transcription of the alginate biosynthesis operon is under tight control by a number of factors, including AmrZ, the focus of this study. Interestingly, multiple transcription factors interact with the far-upstream region of this promoter (PalgD), in which one AmrZ binding site has been identified previously. The mechanisms of AmrZ binding and subsequent activation remain unclear and require more-detailed investigation. In this study, in-depth examinations elucidated four AmrZ binding sites, and their disruption eliminated AmrZ binding and promoter activation. Furthermore, our in vitro fluorescence resonance energy transfer experiments suggest that AmrZ holds together multiple binding sites in PalgD and thereafter induces the formation of higher-order DNA-AmrZ complexes. To determine the importance of interactions between those AmrZ oligomers in the cell, a DNA phasing experiment was performed. PalgD transcription was significantly impaired when the relative phase between AmrZ binding sites was reversed (5 bp), while a full-DNA-turn insertion (10 bp) restored promoter activity. Taken together, the investigations presented here provide a deeper mechanistic understanding of AmrZ-mediated binding to PalgD IMPORTANCE: Overproduction of the exopolysaccharide alginate provides protection to Pseudomonas aeruginosa against antimicrobial treatments and is associated with chronic P. aeruginosa infections in the lungs of cystic fibrosis patients. In this study, we combined a variety of microbiological, genetic, biochemical, and biophysical approaches to investigate the activation of the alginate biosynthesis operon promoter by a key transcription factor named AmrZ. This study has provided important new information on the mechanism of activation of this extremely complex promoter. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The study of zinc ions binding to casein.

PubMed

Pomastowski, P; Sprynskyy, M; Buszewski, B

2014-08-01

The presented research was focused on physicochemical study of casein properties and the kinetics of zinc ions binding to the protein. Moreover, a fast and simple method of casein extraction from cow's milk has been proposed. Casein isoforms, zeta potential (ζ) and particle size of the separated caseins were characterized with the use of capillary electrophoresis, zeta potential analysis and field flow fractionation (FFF) technique, respectively. The kinetics of the metal-binding process was investigated in batch adsorption experiments. Intraparticle diffusion model, first-order and zero-order kinetic models were applied to test the kinetic experimental data. Analysis of changes in infrared bands registered for casein before and after zinc binding was also performed. The obtained results showed that the kinetic process of zinc binding to casein is not homogeneous but is expressed with an initial rapid stage with about 70% of zinc ions immobilized by casein and with a much slower second step. Maximum amount of bound zinc in the experimental conditions was 30.04mgZn/g casein. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular beacons for DNA binding proteins: an emerging technology for detection of DNA binding proteins and their ligands.

PubMed

Dummitt, Benjamin; Chang, Yie-Hwa

2006-06-01

Quantitation of the level or activity of specific proteins is one of the most commonly performed experiments in biomedical research. Protein detection has historically been difficult to adapt to high throughput platforms because of heavy reliance upon antibodies for protein detection. Molecular beacons for DNA binding proteins is a recently developed technology that attempts to overcome such limitations. Protein detection is accomplished using inexpensive, easy-to-synthesize oligonucleotides, accompanied by a fluorescence readout. Importantly, detection of the protein and reporting of the signal occur simultaneously, allowing for one-step protocols and increased potential for use in high throughput analysis. While the initial iteration of the technology allowed only for the detection of sequence-specific DNA binding proteins, more recent adaptations allow for the possibility of development of beacons for any protein, independent of native DNA binding activity. Here, we discuss the development of the technology, the mechanism of the reaction, and recent improvements and modifications made to improve the assay in terms of sensitivity, potential for multiplexing, and broad applicability.

Searching for transcription factor binding sites in vector spaces

PubMed Central

2012-01-01

Background Computational approaches to transcription factor binding site identification have been actively researched in the past decade. Learning from known binding sites, new binding sites of a transcription factor in unannotated sequences can be identified. A number of search methods have been introduced over the years. However, one can rarely find one single method that performs the best on all the transcription factors. Instead, to identify the best method for a particular transcription factor, one usually has to compare a handful of methods. Hence, it is highly desirable for a method to perform automatic optimization for individual transcription factors. Results We proposed to search for transcription factor binding sites in vector spaces. This framework allows us to identify the best method for each individual transcription factor. We further introduced two novel methods, the negative-to-positive vector (NPV) and optimal discriminating vector (ODV) methods, to construct query vectors to search for binding sites in vector spaces. Extensive cross-validation experiments showed that the proposed methods significantly outperformed the ungapped likelihood under positional background method, a state-of-the-art method, and the widely-used position-specific scoring matrix method. We further demonstrated that motif subtypes of a TF can be readily identified in this framework and two variants called the k NPV and k ODV methods benefited significantly from motif subtype identification. Finally, independent validation on ChIP-seq data showed that the ODV and NPV methods significantly outperformed the other compared methods. Conclusions We conclude that the proposed framework is highly flexible. It enables the two novel methods to automatically identify a TF-specific subspace to search for binding sites. Implementations are available as source code at: http://biogrid.engr.uconn.edu/tfbs_search/. PMID:23244338

Valerian extract and valerenic acid are partial agonists of the 5-HT5a receptor in vitro.

PubMed

Dietz, Birgit M; Mahady, Gail B; Pauli, Guido F; Farnsworth, Norman R

2005-08-18

Insomnia is the most frequently encountered sleep complaint worldwide. While many prescription drugs are used to treat insomnia, extracts of valerian (Valeriana officinalis L., Valerianaceae) are also used for the treatment of insomnia and restlessness. To determine novel mechanisms of action, radioligand binding studies were performed with valerian extracts (100% methanol, 50% methanol, dichloromethane [DCM], and petroleum ether [PE]) at the melatonin, glutamate, and GABA(A) receptors, and 8 serotonin receptor subtypes. Both DCM and PE extracts had strong binding affinity to the 5-HT(5a) receptor, but only weak binding affinity to the 5-HT(2b) and the serotonin transporter. Subsequent binding studies focused on the 5-HT(5a) receptor due to the distribution of this receptor in the suprachiasmatic nucleus of the brain, which is implicated in the sleep-wake cycle. The PE extract inhibited [(3)H]lysergic acid diethylamide (LSD) binding to the human 5-HT(5a) receptor (86% at 50 microg/ml) and the DCM extract inhibited LSD binding by 51%. Generation of an IC(50) curve for the PE extract produced a biphasic curve, thus GTP shift experiments were also performed. In the absence of GTP, the competition curve was biphasic (two affinity sites) with an IC(50) of 15.7 ng/ml for the high-affinity state and 27.7 microg/ml for the low-affinity state. The addition of GTP (100 microM) resulted in a right-hand shift of the binding curve with an IC(50) of 11.4 microg/ml. Valerenic acid, the active constituent of both extracts, had an IC(50) of 17.2 microM. These results indicate that valerian and valerenic acid are new partial agonists of the 5-HT(5a) receptor.

Valerian extract and valerenic acid are partial agonists of the 5-HT5a receptor in vitro

PubMed Central

Dietz, Birgit M.; Mahady, Gail B.; Pauli, Guido F.; Farnsworth, Norman R.

2018-01-01

Insomnia is the most frequently encountered sleep complaint worldwide. While many prescription drugs are used to treat insomnia, extracts of valerian (Valeriana officinalis L., Valerianaceae) are also used for the treatment of insomnia and restlessness. To determine novel mechanisms of action, radioligand binding studies were performed with valerian extracts (100% methanol, 50% methanol, dichloromethane [DCM], and petroleum ether [PE]) at the melatonin, glutamate, and GABAA receptors, and 8 serotonin receptor subtypes. Both DCM and PE extracts had strong binding affinity to the 5-HT5a receptor, but only weak binding affinity to the 5-HT2b and the serotonin transporter. Subsequent binding studies focused on the 5-HT5a receptor due to the distribution of this receptor in the suprachiasmatic nucleus of the brain, which is implicated in the sleep–wake cycle. The PE extract inhibited [3H]lysergic acid diethylamide (LSD) binding to the human 5-HT5a receptor (86% at 50 μg/ml) and the DCM extract inhibited LSD binding by 51%. Generation of an IC50 curve for the PE extract produced a biphasic curve, thus GTP shift experiments were also performed. In the absence of GTP, the competition curve was biphasic (two affinity sites) with an IC50 of 15.7 ng/ml for the high-affinity state and 27.7 μg/ml for the low-affinity state. The addition of GTP (100 AM) resulted in a right-hand shift of the binding curve with an IC50 of 11.4 μg/ml. Valerenic acid, the active constituent of both extracts, had an IC50 of 17.2 AM. These results indicate that valerian and valerenic acid are new partial agonists of the 5-HT5a receptor. PMID:15921820

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy*

PubMed Central

Neundlinger, Isabel; Puntheeranurak, Theeraporn; Wildling, Linda; Rankl, Christian; Wang, Lai-Xi; Gruber, Hermann J.; Kinne, Rolf K. H.; Hinterdorfer, Peter

2014-01-01

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2′-aminoethyl β-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations. PMID:24962566

Spatially Resolved Sensitivity of Single-Particle Plasmon Sensors

PubMed Central

2018-01-01

The high sensitivity of localized surface plasmon resonance sensors to the local refractive index allows for the detection of single-molecule binding events. Though binding events of single objects can be detected by their induced plasmon shift, the broad distribution of observed shifts remains poorly understood. Here, we perform a single-particle study wherein single nanospheres bind to a gold nanorod, and relate the observed plasmon shift to the binding location using correlative microscopy. To achieve this we combine atomic force microscopy to determine the binding location, and single-particle spectroscopy to determine the corresponding plasmon shift. As expected, we find a larger plasmon shift for nanospheres binding at the tip of a rod compared to its sides, in good agreement with numerical calculations. However, we also find a broad distribution of shifts even for spheres that were bound at a similar location to the nanorod. Our correlative approach allows us to disentangle effects of nanoparticle dimensions and binding location, and by comparison to numerical calculations we find that the biggest contributor to this observed spread is the dispersion in nanosphere diameter. These experiments provide insight into the spatial sensitivity and signal-heterogeneity of single-particle plasmon sensors and provides a framework for signal interpretation in sensing applications. PMID:29520315

Molecular modeling and SPRi investigations of interleukin 6 (IL6) protein and DNA aptamers.

PubMed

Rhinehardt, Kristen L; Vance, Stephen A; Mohan, Ram V; Sandros, Marinella; Srinivas, Goundla

2018-06-01

Interleukin 6 (IL6), an inflammatory response protein has major implications in immune-related inflammatory diseases. Identification of aptamers for the IL6 protein aids in diagnostic, therapeutic, and theranostic applications. Three different DNA aptamers and their interactions with IL6 protein were extensively investigated in a phosphate buffed saline (PBS) solution. Molecular-level modeling through molecular dynamics provided insights of structural, conformational changes and specific binding domains of these protein-aptamer complexes. Multiple simulations reveal consistent binding region for all protein-aptamer complexes. Conformational changes coupled with quantitative analysis of center of mass (COM) distance, radius of gyration (R g ), and number of intermolecular hydrogen bonds in each IL6 protein-aptamer complex was used to determine their binding performance strength and obtain molecular configurations with strong binding. A similarity comparison of the molecular configurations with strong binding from molecular-level modeling concurred with Surface Plasmon Resonance imaging (SPRi) for these three aptamer complexes, thus corroborating molecular modeling analysis findings. Insights from the natural progression of IL6 protein-aptamer binding modeled in this work has identified key features such as the orientation and location of the aptamer in the binding event. These key features are not readily feasible from wet lab experiments and impact the efficacy of the aptamers in diagnostic and theranostic applications.

Analyzing Ligand Depletion in a Saturation Equilibrium Binding Experiment

ERIC Educational Resources Information Center

Claro, Enrique

2006-01-01

I present a proposal for a laboratory practice to generate and analyze data from a saturation equilibrium binding experiment addressed to advanced undergraduate students. [[superscript 3]H]Quinuclidinyl benzilate is a nonselective muscarinic ligand with very high affinity and very low nonspecific binding to brain membranes, which contain a high…

Theoretical study of the bonding of the first-row transition-metal positive ions to ethylene

NASA Technical Reports Server (NTRS)

Sodupe, M.; Bauschlicher, Charles W., Jr.; Langhoff, Stephen R.; Partridge, Harry

1992-01-01

Ab initio calculations were performed to study the bonding of the first-row transition-metal ions with ethylene. While Sc(+) and Ti(+) insert into the pi bond of ethylene to form a three-membered ring, the ions V(+) through Cu(+) form an electrostatic complex with ethylene. The binding energies are compared with those from experiment and with those of comparable calculations performed previously for the metal-acetylene ion systems.

Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

PubMed

Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

2017-03-17

Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mixed-mode sorption of hydroxylated atrazine degradation products to sell: A mechanism for bound residue

USGS Publications Warehouse

Lerch, R.N.; Thurman, E.M.; Kruger, E.L.

1997-01-01

This study tested the hypothesis that sorption of hydroxylated atrazine degradation products (HADPs: hydroxyatrazine, HA; deethylhydroxyatrazine, DEHA; and deisopropylhydroxyatrazine, DIHA) to soils occurs by mixed-mode binding resulting from two simultaneous mechanisms: (1) cation exchange and (2) hydrophobic interaction. The objective was to use liquid chromatography and soil extraction experiments to show that mixed-mode binding is the mechanism controlling HADP sorption to soils and is also a mechanism for bound residue. Overall, HADP binding to solid-phase extraction (SPE) sorbents occurred in the order: cation exchange >> octadecyl (C18) >> cyanopropyl. Binding to cation exchange SPE and to a high-performance liquid chromatograph octyl (C8) column showed evidence for mixed-mode binding. Comparison of soil extracted by 0.5 M KH2P04, pH 7.5, or 25% aqueous CH3CN showed that, for HA and DIHA, cation exchange was a more important binding mechanism to soils than hydrophobic interaction. Based on differences between several extractants, the extent of HADP mixed-mode binding to soil occurred in the following order: HA > DIHA > DEHA. Mixed-mode extraction recovered 42.8% of bound atrazine residues from aged soil, and 88% of this fraction was identified as HADPs. Thus, a significant portion of bound atrazine residues in soils is sorbed by the mixed-mode binding mechanisms.

Atrazine Molecular Imprinted Polymers: Comparative Analysis by Far-Infrared and Ultraviolet Induced Polymerization

PubMed Central

Chen, Jun; Bai, Lian-Yang; Liu, Kun-Feng; Liu, Run-Qiang; Zhang, Yu-Ping

2014-01-01

Atrazine molecular imprinted polymers (MIPs) were comparatively synthesized using identical polymer formulation by far-infrared (FIR) radiation and ultraviolet (UV)-induced polymerization, respectively. Equilibrium binding experiments were carried out with the prepared MIPs; the results showed that MIPuv possessed specific binding to atrazine compared with their MIPFIR radiation counterparts. Scatchard plot’s of both MIPs indicated that the affinities of the binding sites in MIPs are heterogeneous and can be approximated by two dissociation-constants corresponding to the high-and low-affinity binding sites. Moreover, several common pesticides including atrazine, cyromazine, metamitron, simazine, ametryn, terbutryn were tested to determine their specificity, similar imprinting factor (IF) and different selectivity index (SI) for both MIPs. Physical characterization of the polymers revealed that the different polymerization methods led to slight differences in polymer structures and performance by scanning electron microscope (SEM), Fourier transform infrared absorption (FT-IR), and mercury analyzer (MA). Finally, both MIPs were used as selective sorbents for solid phase extraction (SPE) of atrazine from lake water, followed by high performance liquid chromatography (HPLC) analysis. Compared with commercial C18 SPE sorbent (86.4%–94.8%), higher recoveries of atrazine in spiked lake water were obtained in the range of 90.1%–97.1% and 94.4%–101.9%, for both MIPs, respectively. PMID:24398982

Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA

NASA Astrophysics Data System (ADS)

Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

2016-01-01

A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.

Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA.

PubMed

Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

2016-01-15

A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging. Copyright © 2015 Elsevier B.V. All rights reserved.

Solution structure and interactions of the Escherichia coli cell division activator protein CedA.

PubMed

Chen, Ho An; Simpson, Peter; Huyton, Trevor; Roper, David; Matthews, Stephen

2005-05-10

CedA is a protein that is postulated to be involved in the regulation of cell division in Escherichia coli and related organisms; however, little biological data about its possible mode of action are available. Here we present a three-dimensional structure of this protein as determined by NMR spectroscopy. The protein is made up of four antiparallel beta-strands, an alpha-helix, and a large unstructured stretch of residues at the N-terminus. It shows structural similarity to a family of DNA-binding proteins which interact with dsDNA via a three-stranded beta-sheet, suggesting that CedA may be a DNA-binding protein. The putative binding surface of CedA is predominantly positively charged with a number of basic residues surrounding a groove largely dominated by aromatic residues. NMR chemical shift perturbations and gel-shift experiments performed with CedA confirm that the protein binds dsDNA, and its interaction is mediated primarily via the beta-sheet.

GPU-Based Point Cloud Superpositioning for Structural Comparisons of Protein Binding Sites.

PubMed

Leinweber, Matthias; Fober, Thomas; Freisleben, Bernd

2018-01-01

In this paper, we present a novel approach to solve the labeled point cloud superpositioning problem for performing structural comparisons of protein binding sites. The solution is based on a parallel evolution strategy that operates on large populations and runs on GPU hardware. The proposed evolution strategy reduces the likelihood of getting stuck in a local optimum of the multimodal real-valued optimization problem represented by labeled point cloud superpositioning. The performance of the GPU-based parallel evolution strategy is compared to a previously proposed CPU-based sequential approach for labeled point cloud superpositioning, indicating that the GPU-based parallel evolution strategy leads to qualitatively better results and significantly shorter runtimes, with speed improvements of up to a factor of 1,500 for large populations. Binary classification tests based on the ATP, NADH, and FAD protein subsets of CavBase, a database containing putative binding sites, show average classification rate improvements from about 92 percent (CPU) to 96 percent (GPU). Further experiments indicate that the proposed GPU-based labeled point cloud superpositioning approach can be superior to traditional protein comparison approaches based on sequence alignments.

Zonal Rate Model for Axial and Radial Flow Membrane Chromatography. Part I: Knowledge Transfer Across Operating Conditions and Scales

PubMed Central

Ghosh, Pranay; Vahedipour, Kaveh; Lin, Min; Vogel, Jens H; Haynes, Charles A; von Lieres, Eric

2013-01-01

The zonal rate model (ZRM) has previously been applied for analyzing the performance of axial flow membrane chromatography capsules by independently determining the impacts of flow and binding related non-idealities on measured breakthrough curves. In the present study, the ZRM is extended to radial flow configurations, which are commonly used at larger scales. The axial flow XT5 capsule and the radial flow XT140 capsule from Pall are rigorously analyzed under binding and non-binding conditions with bovine serum albumin (BSA) as test molecule. The binding data of this molecule is much better reproduced by the spreading model, which hypothesizes different binding orientations, than by the well-known Langmuir model. Moreover, a revised cleaning protocol with NaCl instead of NaOH and minimizing the storage time has been identified as most critical for quantitatively reproducing the measured breakthrough curves. The internal geometry of both capsules is visualized by magnetic resonance imaging (MRI). The flow in the external hold-up volumes of the XT140 capsule was found to be more homogeneous as in the previously studied XT5 capsule. An attempt for model-based scale-up was apparently impeded by irregular pleat structures in the used XT140 capsule, which might lead to local variations in the linear velocity through the membrane stack. However, the presented approach is universal and can be applied to different capsules. The ZRM is shown to potentially help save valuable material and time, as the experiments required for model calibration are much cheaper than the predicted large-scale experiment at binding conditions. Biotechnol. Bioeng. 2013; 110: 1129–1141. © 2012 Wiley Periodicals, Inc. PMID:23097218

Working Memory and Aging: Separating the Effects of Content and Context

PubMed Central

Bopp, Kara L.; Verhaeghen, Paul

2009-01-01

In three experiments, we investigated the hypothesis that age-related differences in working memory might be due to the inability to bind content with context. Participants were required to find a repeating stimulus within a single series (no context memory required) or within multiple series (necessitating memory for context). Response time and accuracy were examined in two task domains: verbal and visuospatial. Binding content with context led to longer processing time and poorer accuracy in both age groups, even when working memory load was held constant. Although older adults were overall slower and less accurate than younger adults, the need for context memory did not differentially affect their performance. It is therefore unlikely that age differences in working memory are due to specific age-related problems with content-with-context binding. PMID:20025410

Towards water compatible MIPs for sensing in aqueous media.

PubMed

Horemans, F; Weustenraed, A; Spivak, D; Cleij, T J

2012-06-01

When synthesizing molecularly imprinted polymers (MIPs), a few fundamental principles should be kept in mind. There is a strong correlation between porogen polarity, MIP microenvironment polarity and the imprinting effect itself. The combination of these parameters eventually determines the overall binding behavior of a MIP in a given solvent. In addition, it is shown that MIP binding is strongly influenced by the polarity of the rebinding solvent. Because the use of MIPs in biomedical environments is of considerable interest, it is important that these MIPs perform well in aqueous media. In this article, various approaches are explored towards a water compatible MIP for the target molecule l-nicotine. To this end, the imprinting effect together with the MIP matrix polarity is fine-tuned during MIP synthesis. The binding behavior of the resulting MIPs is evaluated by performing batch rebinding experiments that makes it possible to select the most suitable MIP/non-imprinted polymer couple for future application in aqueous environments. One method to achieve improved compatibility with water is referred to as porogen tuning, in which porogens of varying polarities are used. It is demonstrated that, especially when multiple porogens are mixed, this approach can lead to superior performance in aqueous environments. Another method involves the incorporation of polar or non-polar comonomers in the MIP matrix. It is shown that by carefully selecting these monomers, it is also possible to obtain MIPs, which can selectively bind their target in water. Copyright © 2012 John Wiley & Sons, Ltd.

Binding energies and spatial structures of small carrier complexes in monolayer transition-metal dichalcogenides via diffusion Monte Carlo

DOE PAGES

Mayers, Matthew Z.; Berkelbach, Timothy C.; Hybertsen, Mark S.; ...

2015-10-09

Ground-state diffusion Monte Carlo is used to investigate the binding energies and intercarrier radial probability distributions of excitons, trions, and biexcitons in a variety of two-dimensional transition-metal dichalcogenide materials. We compare these results to approximate variational calculations, as well as to analogous Monte Carlo calculations performed with simplified carrier interaction potentials. Our results highlight the successes and failures of approximate approaches as well as the physical features that determine the stability of small carrier complexes in monolayer transition-metal dichalcogenide materials. In conclusion, we discuss points of agreement and disagreement with recent experiments.

Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

NASA Astrophysics Data System (ADS)

Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

1998-01-01

The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

Investigation of glucose binding sites on insulin.

PubMed

Zoete, Vincent; Meuwly, Markus; Karplus, Martin

2004-05-15

Possible insulin binding sites for D-glucose have been investigated theoretically by docking and molecular dynamics (MD) simulations. Two different docking programs for small molecules were used; Multiple Copy Simultaneous Search (MCSS) and Solvation Energy for Exhaustive Docking (SEED) programs. The configurations resulting from the MCSS search were evaluated with a scoring function developed to estimate the binding free energy. SEED calculations were performed using various values for the dielectric constant of the solute. It is found that scores emphasizing non-polar interactions gave a preferential binding site in agreement with that inferred from recent fluorescence and NMR NOESY experiments. The calculated binding affinity of -1.4 to -3.5 kcal/mol is within the measured range of -2.0 +/- 0.5 kcal/mol. The validity of the binding site is suggested by the dynamical stability of the bound glucose when examined with MD simulations with explicit solvent. Alternative binding sites were found in the simulations and their relative stabilities were estimated. The motions of the bound glucose during molecular dynamics simulations are correlated with the motions of the insulin side chains that are in contact with it and with larger scale insulin motions. These results raise the question of whether glucose binding to insulin could play a role in its activity. The results establish the complementarity of molecular dynamics simulations and normal mode analyses with the search for binding sites proposed with small molecule docking programs. Copyright 2004 Wiley-Liss, Inc.

Antagonism of human CC-chemokine receptor 4 can be achieved through three distinct binding sites on the receptor

PubMed Central

Slack, Robert J; Russell, Linda J; Barton, Nick P; Weston, Cathryn; Nalesso, Giovanna; Thompson, Sally-Anne; Allen, Morven; Chen, Yu Hua; Barnes, Ashley; Hodgson, Simon T; Hall, David A

2013-01-01

Chemokine receptor antagonists appear to access two distinct binding sites on different members of this receptor family. One class of CCR4 antagonists has been suggested to bind to a site accessible from the cytoplasm while a second class did not bind to this site. In this report, we demonstrate that antagonists representing a variety of structural classes bind to two distinct allosteric sites on CCR4. The effects of pairs of low-molecular weight and/or chemokine CCR4 antagonists were evaluated on CCL17- and CCL22-induced responses of human CCR4+ T cells. This provided an initial grouping of the antagonists into sets which appeared to bind to distinct binding sites. Binding studies were then performed with radioligands from each set to confirm these groupings. Some novel receptor theory was developed to allow the interpretation of the effects of the antagonist combinations. The theory indicates that, generally, the concentration-ratio of a pair of competing allosteric modulators is maximally the sum of their individual effects while that of two modulators acting at different sites is likely to be greater than their sum. The low-molecular weight antagonists could be grouped into two sets on the basis of the functional and binding experiments. The antagonistic chemokines formed a third set whose behaviour was consistent with that of simple competitive antagonists. These studies indicate that there are two allosteric regulatory sites on CCR4. PMID:25505571

More insight into the interplay of response selection and visual attention in dual-tasks: masked visual search and response selection are performed in parallel.

PubMed

Reimer, Christina B; Schubert, Torsten

2017-09-15

Both response selection and visual attention are limited in capacity. According to the central bottleneck model, the response selection processes of two tasks in a dual-task situation are performed sequentially. In conjunction search, visual attention is required to select the items and to bind their features (e.g., color and form), which results in a serial search process. Search time increases as items are added to the search display (i.e., set size effect). When the search display is masked, visual attention deployment is restricted to a brief period of time and target detection decreases as a function of set size. Here, we investigated whether response selection and visual attention (i.e., feature binding) rely on a common or on distinct capacity limitations. In four dual-task experiments, participants completed an auditory Task 1 and a conjunction search Task 2 that were presented with an experimentally modulated temporal interval between them (Stimulus Onset Asynchrony, SOA). In Experiment 1, Task 1 was a two-choice discrimination task and the conjunction search display was not masked. In Experiment 2, the response selection difficulty in Task 1 was increased to a four-choice discrimination and the search task was the same as in Experiment 1. We applied the locus-of-slack method in both experiments to analyze conjunction search time, that is, we compared the set size effects across SOAs. Similar set size effects across SOAs (i.e., additive effects of SOA and set size) would indicate sequential processing of response selection and visual attention. However, a significantly smaller set size effect at short SOA compared to long SOA (i.e., underadditive interaction of SOA and set size) would indicate parallel processing of response selection and visual attention. In both experiments, we found underadditive interactions of SOA and set size. In Experiments 3 and 4, the conjunction search display in Task 2 was masked. Task 1 was the same as in Experiments 1 and 2, respectively. In both experiments, the d' analysis revealed that response selection did not affect target detection. Overall, Experiments 1-4 indicated that neither the response selection difficulty in the auditory Task 1 (i.e., two-choice vs. four-choice) nor the type of presentation of the search display in Task 2 (i.e., not masked vs. masked) impaired parallel processing of response selection and conjunction search. We concluded that in general, response selection and visual attention (i.e., feature binding) rely on distinct capacity limitations.

BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP.

PubMed

Schneider, Markus; Rosam, Mathias; Glaser, Manuel; Patronov, Atanas; Shah, Harpreet; Back, Katrin Christiane; Daake, Marina Angelika; Buchner, Johannes; Antes, Iris

2016-10-01

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Tyrosine411 and Arginine410 of Human Serum Albumin Play an Important Role in the Binding of Sodium 4-Phenylbutyrate to Site II.

PubMed

Enokida, Taisuke; Yamasaki, Keishi; Okamoto, Yuko; Taguchi, Kazuaki; Ishiguro, Takako; Maruyama, Toru; Seo, Hakaru; Otagiri, Masaki

2016-06-01

Sodium 4-phenylbutyrate (PB) has many pharmacological activities; therefore extending its clinical use to the treatment of a wider variety of diseases would be desirable. However, our knowledge of the binding of PB to plasma proteins is not extensive. To address this issue in more detail, we characterized the protein binding of PB. Binding experiments showed that PB mainly binds to human serum albumin (HSA) in plasma. PB was also found to bind to a single site on HSA, which was identified as site II by fluorescent probe displacement experiment. Furthermore, an appropriate alkyl chain length and a carboxylic group in the PB structure were required for PB binding to HSA, suggesting that hydrophobic (and van der Waals) and electrostatic interactions are involved as binding modes. The contributions of hydrogen bonding and/or van der Waals interactions were also indicated by thermodynamic analyses. Tyrosine411 and arginine410 were identified as being involved in the binding of PB to site II, based on binding experiments using chemically modified- and mutant-HSA preparations. In conclusion, the available evidence indicates that PB binds to site II of HSA with assistance by multiple forces and that tyrosine411 and arginine410 both play important roles in this phenomenon. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Selective binding of lectins to normal and neoplastic urothelium in rat and mouse bladder carcinogenesis models.

PubMed

Zupančič, Daša; Kreft, Mateja Erdani; Romih, Rok

2014-01-01

Bladder cancer adjuvant intravesical therapy could be optimized by more selective targeting of neoplastic tissue via specific binding of lectins to plasma membrane carbohydrates. Our aim was to establish rat and mouse models of bladder carcinogenesis to investigate in vivo and ex vivo binding of selected lectins to the luminal surface of normal and neoplastic urothelium. Male rats and mice were treated with 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water and used for ex vivo and in vivo lectin binding experiments. Urinary bladder samples were also used for paraffin embedding, scanning electron microscopy and immunofluorescence labelling of uroplakins. During carcinogenesis, the structure of the urinary bladder luminal surface changed from microridges to microvilli and ropy ridges and the expression of urothelial-specific glycoproteins uroplakins was decreased. Ex vivo and in vivo lectin binding experiments gave comparable results. Jacalin (lectin from Artocarpus integrifolia) exhibited the highest selectivity for neoplastic compared to normal urothelium of rats and mice. The binding of lectin from Amaranthus caudatus decreased in rat model and increased in mouse carcinogenesis model, indicating interspecies variations of plasma membrane glycosylation. Lectin from Datura stramonium showed higher affinity for neoplastic urothelium compared to the normal in rat and mouse model. The BBN-induced animal models of bladder carcinogenesis offer a promising approach for lectin binding experiments and further lectin-mediated targeted drug delivery research. Moreover, in vivo lectin binding experiments are comparable to ex vivo experiments, which should be considered when planning and optimizing future research.

A heteronuclear zero quantum coherence Nz-exchange experiment that resolves resonance overlap and its application to measure the rates of heme binding to the IsdC protein.

PubMed

Robson, Scott A; Peterson, Robert; Bouchard, Louis-S; Villareal, Valerie A; Clubb, Robert T

2010-07-21

Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or N(z)-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein-ligand binding equilibria for (15)N nuclei. To overcome the problem of (15)N chemical shift degeneracy we developed a heteronuclear zero-quantum (and double-quantum) coherence N(z)-exchange experiment that resolves (15)N chemical shift degeneracy in the indirect dimension. We demonstrate the utility of this new experiment by measuring the heme binding kinetics of the IsdC protein from Staphylococcus aureus. Because of peak overlap, we could not reliably analyze binding kinetics using conventional methods. However, our new experiment resulted in six well-resolved systems that yielded interpretable data. We measured a relatively slow k(off) rate of heme from IsdC (<10 s(-1)), which we interpret as necessary so heme loaded IsdC has time to encounter downstream binding partners to which it passes the heme. The utility of using this new exchange experiment can be easily expanded to (13)C nuclei. We expect our heteronuclear zero-quantum coherence N(z)-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.

Binding biological motion and visual features in working memory.

PubMed

Ding, Xiaowei; Zhao, Yangfan; Wu, Fan; Lu, Xiqian; Gao, Zaifeng; Shen, Mowei

2015-06-01

Working memory mechanisms for binding have been examined extensively in the last decade, yet few studies have explored bindings relating to human biological motion (BM). Human BM is the most salient and biologically significant kinetic information encountered in everyday life and is stored independently from other visual features (e.g., colors). The current study explored 3 critical issues of BM-related binding in working memory: (a) how many BM binding units can be retained in working memory, (b) whether involuntarily object-based binding occurs during BM binding, and (c) whether the maintenance of BM bindings in working memory requires attention above and beyond that needed to maintain the constituent dimensions. We isolated motion signals of human BM from non-BM sources by using point-light displays as to-be-memorized BM and presented the participants colored BM in a change detection task. We found that working memory capacity for BM-color bindings is rather low; only 1 or 2 BM-color bindings could be retained in working memory regardless of the presentation manners (Experiments 1-3). Furthermore, no object-based encoding took place for colored BM stimuli regardless of the processed dimensions (Experiments 4 and 5). Central executive attention contributes to the maintenance of BM-color bindings, yet maintaining BM bindings in working memory did not require more central attention than did maintaining the constituent dimensions in working memory (Experiment 6). Overall, these results suggest that keeping BM bindings in working memory is a fairly resource-demanding process, yet central executive attention does not play a special role in this cross-module binding. (c) 2015 APA, all rights reserved).

Allosteric regulation of focal adhesion kinase by PIP₂ and ATP.

PubMed

Zhou, Jing; Bronowska, Agnieszka; Le Coq, Johanne; Lietha, Daniel; Gräter, Frauke

2015-02-03

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains--the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP₂) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP₂. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP₂. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP₂ binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP₂ to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP₂ binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

NMR spectroscopy of the ligand binding core of ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists

PubMed Central

Fenwick, Michael K.; Oswald, Robert E.

2008-01-01

Glutamate receptors mediate neuronal intercommunication in the central nervous system by coupling extracellular neurotransmitter-receptor interactions to ion channel conductivity. To gain insight into structural and dynamical factors that underlie this coupling, solution NMR experiments were performed on the bi-lobed ligand-binding core of glutamate receptor 2 in complexes with a set of willardiine partial agonists. These agonists are valuable for studying structure-function relationships because their 5-position substituent size is correlated with ligand efficacy and extent of receptor desensitization whereas the substituent electronegativity is correlated with ligand potency. NMR results show that the protein backbone amide chemical shift deviations correlate mainly with efficacy and extent of desensitization. Pronounced deviations occur at specific residues in the ligand-binding site and in the two helical segments that join the lobes by a disulfide bond. Experiments detecting conformational exchange show that micro- to millisecond timescale motions also occur near the disulfide bond and vary largely with efficacy and extent of desensitization. These results thus identify regions displaying structural and dynamical dissimilarity arising from differences in ligand-protein interactions and lobe closure which may play a critical role in receptor response. Furthermore, measures of line broadening and conformational exchange for a portion of the ligand-binding site correlate with ligand EC50 data. These results do not have any correlate in the currently available crystal structures and thus provide a novel view of ligand-binding events that may be associated with agonist potency differences. PMID:18387631

Highly accessible AU-rich regions in 3' untranslated regions are hotspots for binding of regulatory factors.

PubMed

Plass, Mireya; Rasmussen, Simon H; Krogh, Anders

2017-04-01

Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3'UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing "free" target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer of regulation of post-transcriptional regulatory networks.

Highly accessible AU-rich regions in 3’ untranslated regions are hotspots for binding of regulatory factors

PubMed Central

2017-01-01

Post-transcriptional regulation is regarded as one of the major processes involved in the regulation of gene expression. It is mainly performed by RNA binding proteins and microRNAs, which target RNAs and typically affect their stability. Recent efforts from the scientific community have aimed at understanding post-transcriptional regulation at a global scale by using high-throughput sequencing techniques such as cross-linking and immunoprecipitation (CLIP), which facilitates identification of binding sites of these regulatory factors. However, the diversity in the experimental procedures and bioinformatics analyses has hindered the integration of multiple datasets and thus limited the development of an integrated view of post-transcriptional regulation. In this work, we have performed a comprehensive analysis of 107 CLIP datasets from 49 different RBPs in HEK293 cells to shed light on the complex interactions that govern post-transcriptional regulation. By developing a more stringent CLIP analysis pipeline we have discovered the existence of conserved regulatory AU-rich regions in the 3’UTRs where miRNAs and RBPs that regulate several processes such as polyadenylation or mRNA stability bind. Analogous to promoters, many factors have binding sites overlapping or in close proximity in these hotspots and hence the regulation of the mRNA may depend on their relative concentrations. This hypothesis is supported by RBP knockdown experiments that alter the relative concentration of RBPs in the cell. Upon AGO2 knockdown (KD), transcripts containing “free” target sites show increased expression levels compared to those containing target sites in hotspots, which suggests that target sites within hotspots are less available for miRNAs to bind. Interestingly, these hotspots appear enriched in genes with regulatory functions such as DNA binding and RNA binding. Taken together, our results suggest that hotspots are functional regulatory elements that define an extra layer of regulation of post-transcriptional regulatory networks. PMID:28410363

Effect of solid surface charge on the binding behaviour of a metal-binding peptide

PubMed Central

Donatan, Senem; Sarikaya, Mehmet; Tamerler, Candan; Urgen, Mustafa

2012-01-01

Over the last decade, solid-binding peptides have been increasingly used as molecular building blocks coupling bio- and nanotechnology. Despite considerable research being invested in this field, the effects of many surface-related parameters that define the binding of peptide to solids are still unknown. In the quest to control biological molecules at solid interfaces and, thereby, tailoring the binding characteristics of the peptides, the use of surface charge of the solid surface may probably play an important role, which then can be used as a potential tuning parameter of peptide adsorption. Here, we report quantitative investigation on the viscoelastic properties and binding kinetics of an engineered gold-binding peptide, 3RGBP1, adsorbed onto the gold surface at different surface charge densities. The experiments were performed in aqueous solutions using an electrochemical dissipative quartz crystal microbalance system. Hydrodynamic mass, hydration state and surface coverage of the adsorbed peptide films were determined as a function of surface charge density of the gold metal substrate. Under each charged condition, binding of 3rGBP1 displayed quantitative differences in terms of adsorbed peptide amount, surface coverage ratio and hydration state. Based on the intrinsically disordered structure of the peptide, we propose a possible mechanism for binding of the peptide that can be used for tuning surface adsorption in further studies. Controlled alteration of peptide binding on solid surfaces, as shown here, may provide novel methods for surface functionalization used for bioenabled processing and fabrication of future micro- and nanodevices. PMID:22491974

The effect of gamma-enhancing binaural beats on the control of feature bindings.

PubMed

Colzato, Lorenza S; Steenbergen, Laura; Sellaro, Roberta

2017-07-01

Binaural beats represent the auditory experience of an oscillating sound that occurs when two sounds with neighboring frequencies are presented to one's left and right ear separately. Binaural beats have been shown to impact information processing via their putative role in increasing neural synchronization. Recent studies of feature-repetition effects demonstrated interactions between perceptual features and action-related features: repeating only some, but not all features of a perception-action episode hinders performance. These partial-repetition (or binding) costs point to the existence of temporary episodic bindings (event files) that are automatically retrieved by repeating at least one of their features. Given that neural synchronization in the gamma band has been associated with visual feature bindings, we investigated whether the impact of binaural beats extends to the top-down control of feature bindings. Healthy adults listened to gamma-frequency (40 Hz) binaural beats or to a constant tone of 340 Hz (control condition) for ten minutes before and during a feature-repetition task. While the size of visuomotor binding costs (indicating the binding of visual and action features) was unaffected by the binaural beats, the size of visual feature binding costs (which refer to the binding between the two visual features) was considerably smaller during gamma-frequency binaural beats exposure than during the control condition. Our results suggest that binaural beats enhance selectivity in updating episodic memory traces and further strengthen the hypothesis that neural activity in the gamma band is critically associated with the control of feature binding.

Hyper-binding: a unique age effect.

PubMed

Campbell, Karen L; Hasher, Lynn; Thomas, Ruthann C

2010-03-01

Previous work has shown that older adults encode lexical and semantic information about verbal distractors and use that information to facilitate performance on subsequent tasks. In this study, we investigated whether older adults also form associations between distractors and co-occurring targets. In two experiments, participants performed a 1-back task on pictures superimposed with irrelevant words; 10 min later, participants were given a paired-associates memory task without reference to the 1-back task. The study list included preserved and re-paired (disrupted) pairs from the 1-back task. Older adults showed a memory advantage for preserved pairs and a disadvantage for disrupted pairs, whereas younger adults performed similarly across pair types. These results suggest the existence of a hyper-binding phenomenon in which older adults encode seemingly extraneous co-occurrences in the environment and transfer this knowledge to subsequent tasks. This increased knowledge of how events covary may be the reason why real-world decision-making ability is retained, or even enhanced, with age.

Competitive binding experiments can reduce the false positive results of affinity-based ultrafiltration-HPLC: A case study for identification of potent xanthine oxidase inhibitors from Perilla frutescens extract.

PubMed

Wang, Zhiqiang; Kwon, Shin Hwa; Hwang, Seung Hwan; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

2017-03-24

The purpose of this study was to assess the possibility of using competitive binding experiments with ultrafiltration-HPLC analysis to identify potent xanthine oxidase (XO) inhibitors from the Perilla frutescens extract as an attempt to reduce the number of false positive results. To isolate the enzyme-ligand complex from unbound compounds, the P. frutescens extract was either incubated in the absence of XO, in the presence of XO, or with the active site blocked XO before the ultrafiltration was performed. Allopurinaol was used as the XO active site blocker. The unbound compounds were subjected to HPLC analysis. The degree of total binding (TBD) and degree of specific binding (SBD) of each compound were calculated using the peak areas. TBD represents the binding affinities of compounds from the P. frutescens extract for the XO binding site. SBD represents the XO competitive binding between allopurinol and ligands from the extract samples. Two criteria were applied to select putative targets that could help avoid false positives. These include TBD>30% and SBD>10%. Using that approach, kaempferol-3-O-rutinoside, rosmarinic acid, methyl-rosmarinic acid, apigenin, and 4',5,7-trimethoxyflavone were identified, from total 11 compounds, as potent XO inhibitors. Finally, apigenin, 4',5,7-trimethoxyflavone, and luteolin were XO inhibitors verified through an XO inhibition assay and structural simulation of the complex. These results showed that the newly developed strategy has the advantage that the number of targets identified via ultrafiltration-HPLC can be narrowed from many false positives. However, not all false positives can be eliminated with this approach. Some potent inhibitors might also be excluded with the use of this method. The limitations of this method are also discussed herein. Copyright © 2017 Elsevier B.V. All rights reserved.

Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations.

PubMed

Thompson, Damien; Lazennec, Christine; Plateau, Pierre; Simonson, Thomas

2008-05-15

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand. 2007 Wiley-Liss, Inc.

Fluorescence Microscopy of Nanochannel-Confined DNA.

PubMed

Westerlund, Fredrik; Persson, Fredrik; Fritzsche, Joachim; Beech, Jason P; Tegenfeldt, Jonas O

2018-01-01

Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level and both the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments and analyze the data.

Kinetic method for the large-scale analysis of the binding mechanism of histone deacetylase inhibitors.

PubMed

Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

2014-09-01

Performing kinetic studies on protein ligand interactions provides important information on complex formation and dissociation. Beside kinetic parameters such as association rates and residence times, kinetic experiments also reveal insights into reaction mechanisms. Exploiting intrinsic tryptophan fluorescence a parallelized high-throughput Förster resonance energy transfer (FRET)-based reporter displacement assay with very low protein consumption was developed to enable the large-scale kinetic characterization of the binding of ligands to recombinant human histone deacetylases (HDACs) and a bacterial histone deacetylase-like amidohydrolase (HDAH) from Bordetella/Alcaligenes. For the binding of trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and two other SAHA derivatives to HDAH, two different modes of action, simple one-step binding and a two-step mechanism comprising initial binding and induced fit, were verified. In contrast to HDAH, all compounds bound to human HDAC1, HDAC6, and HDAC8 through a two-step mechanism. A quantitative view on the inhibitor-HDAC systems revealed two types of interaction, fast binding and slow dissociation. We provide arguments for the thesis that the relationship between quantitative kinetic and mechanistic information and chemical structures of compounds will serve as a valuable tool for drug optimization. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparison of the interaction between lactoferrin and isomeric drugs

NASA Astrophysics Data System (ADS)

Guo, Ming; Lu, Xiaowang; Wang, Yan; Brodelius, Peter E.

2017-02-01

The binding properties of pentacyclic triterpenoid isomeric drugs, i.e. ursolic acid (UA) and oleanolic acid (OA), to bovine lactoferrin (BLF) have been studied by molecule modeling, fluorescence spectroscopy, UV-visible absorbance spectroscopy and infrared spectroscopy (IR). Molecular docking, performed to reveal the possible binding mode or mechanism, suggested that hydrophobic interaction and hydrogen bonding play important roles to stabilize the complex. The results of spectroscopic measurements showed that the two isomeric drugs both strongly quenched the intrinsic fluorescence of BLF through a static quenching procedure although some differences between UA and OA binding strength and non-radiation energy transfer occurred within the molecules. The number of binding sites was 3.44 and 3.10 for UA and OA, respectively, and the efficiency of Förster energy transfer provided a distance of 0.77 and 1.21 nm for UA and OA, respectively. The conformation transformation of BLF affected by the drugs conformed to the ;all-or-none; pattern. In addition, the changes of the ratios of α-helices, β-sheets and β-turns of BLF during the process of the interaction were obtained. The results of the experiments in combination with the calculations showed that there are two modes of pentacyclic triterpenoid binding to BLF instead of one binding mode only governed by the principle of the lowest bonding energy.

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

NASA Astrophysics Data System (ADS)

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

2015-11-01

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

CompGO: an R package for comparing and visualizing Gene Ontology enrichment differences between DNA binding experiments.

PubMed

Waardenberg, Ashley J; Basset, Samuel D; Bouveret, Romaric; Harvey, Richard P

2015-09-02

Gene ontology (GO) enrichment is commonly used for inferring biological meaning from systems biology experiments. However, determining differential GO and pathway enrichment between DNA-binding experiments or using the GO structure to classify experiments has received little attention. Herein, we present a bioinformatics tool, CompGO, for identifying Differentially Enriched Gene Ontologies, called DiEGOs, and pathways, through the use of a z-score derivation of log odds ratios, and visualizing these differences at GO and pathway level. Through public experimental data focused on the cardiac transcription factor NKX2-5, we illustrate the problems associated with comparing GO enrichments between experiments using a simple overlap approach. We have developed an R/Bioconductor package, CompGO, which implements a new statistic normally used in epidemiological studies for performing comparative GO analyses and visualizing comparisons from . BED data containing genomic coordinates as well as gene lists as inputs. We justify the statistic through inclusion of experimental data and compare to the commonly used overlap method. CompGO is freely available as a R/Bioconductor package enabling easy integration into existing pipelines and is available at: http://www.bioconductor.org/packages/release/bioc/html/CompGO.html packages/release/bioc/html/CompGO.html.

The IκBα/NF-κB complex has two hot spots, one at either end of the interface

PubMed Central

Bergqvist, Simon; Ghosh, Gourisankar; Komives, Elizabeth A.

2008-01-01

IκBα binds to and inhibits the transcriptional activity of NF-κB family members via its ankyrin repeat (AR) domain. The binding affinity of IκBα with NF-κB(p50/p65) heterodimers and NF-κB(p65/65) homodimers is in the picomolar range, and in the cell, this results in long half-lives of the complexes. Direct binding experiments have been performed using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) on a series of truncations and mutations in order to understand what regions of the interface are most important for the tight binding affinity of this complex. We previously showed that interactions between residues 305 and 321 of NF-κB(p65) with the first AR of IκBα are critical for the binding energy. Interactions in this region are responsible for more than 7 kcal/mol of the binding energy. Here we show equally drastic consequences for the binding energy occur upon truncation of even a few residues at the C terminus of IκBα. Thus, the interface actually has two hot spots, one at either end of the elongated and large surface of interaction. These results suggest a “squeeze” mechanism that leads to the extremely high affinity of the IκBα•NF-κB complex through stabilization of the ankyrin repeat domain. PMID:18824506

Spectroscopic analysis of the interaction between thiazolo[2,3-b]pyrimidine analogues and bovine serum albumin.

PubMed

Yu, Xianyong; Yang, Ying; Yao, Qing; Tao, Hongwen; Lu, Shiyu; Xie, Jian; Zhou, Hu; Yi, Pinggui

2012-10-01

The interaction between thiazolo[2,3-b]pyrimidine (TZPM) analogues and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy and UV-Vis spectroscopy at two different temperatures (299 and 307K) under imitated physiological conditions. The results indicate that both static quenching and dynamic quenching contribute to the fluorescence quenching of BSA by TZPM. The binding constant (K(a)) and binding sites (n) were calculated from the obtained spectra. Based on the Förster non-radiation energy transfer theory, the average binding distance between BSA and TZPM was estimated. The synchronous fluorescence spectra indicate that the conformation of BSA has been changed. The comparison of binding potency of TZPM and BSA suggests that the substituents on the benzene ring enhance the binding affinity of TZPM and BSA. We investigated the possible sub-domains on BSA that bind TZPM by displacement experiments. Furthermore, to explore the effect of molecular structure on the binding, a study on quantitative structure-property relationship (QSPR) was performed, the quantitative relationship equation of R(0), r and K(a) were obtained. We observed that R(0), r and K(a) between BSA and TZPM is connected with the margin of the highest and the lowest occupied orbital energy (ΔE), dipole moment (μ), Molar Volume (V(m)), Mole Mass (M). Copyright © 2012 Elsevier B.V. All rights reserved.

Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10

PubMed Central

Russ, Ulrich; Metzger, Friedrich; Kickenweiz, Elisabeth; Hambrock, Annette; Krippeit-Drews, Peter; Quast, Ulrich

1997-01-01

The ATP-sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]-P1075, and by electrophysiological techniques. Saturation binding experiments gave a KD value of 9.2±5.2 nM and a binding capacity (BMax) of 140±40 fmol mg−1 protein for [3H]-P1075 binding to A10 cells; from the BMax value a density of binding sites of 5–10 per μm2 plasmalemma was estimated. KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. Resting membrane potential, recorded with microelectrodes, was −51±1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to −25 mV with EC50 values of 170±40 nM and 870±190 nM, respectively. The hyperpolarization induced by levcromakalim (3 μM) was completely reversed by glibenclamide with an IC50 value of 86±17 nM. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 μM) induced a current which reversed around −80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 μM) abolished the effect of levcromakalim. Analysis of the noise of the levcromakalim (10 μM)-induced current at −40 and −20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 μm−2, 8.8 pS and 0.39, respectively. The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells. PMID:9401776

Synthesis and crystal structure determination of copper(II)-complex: In vitro DNA and HSA binding, pBR322 plasmid cleavage, cell imaging and cytotoxic studies.

PubMed

Tabassum, Sartaj; Zaki, Mehvash; Ahmad, Musheer; Afzal, Mohd; Srivastav, Saurabh; Srikrishna, Saripella; Arjmand, Farukh

2014-08-18

New Cu(II) complex 1 of indole-3-propionic acid and 1,10-phenanthroline was synthesized and characterized by analytical, spectroscopic and single crystal X-ray diffraction. In vitro DNA binding studies of 1 was performed by employing UV-vis and fluorescence spectroscopic techniques. The binding affinity towards human serum albumin (HSA) was also investigated to understand the carrier role in body system, as the time dependent HPLC experiment of 1 revealed that bonded drug with protein releases slowly in presence of DNA. Complex 1 exhibited good anti-tumor activity (GI50 values <10 μg/ml), and to elucidate the mechanism of tumor inhibition, topoisomerase I enzymatic activity was carried out and further validated by cell imaging studies which clearly showed its nuclear localization. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Interaction of phenazinium dyes with double-stranded poly(A): Spectroscopy and isothermal titration calorimetry studies

NASA Astrophysics Data System (ADS)

Khan, Asma Yasmeen; Saha, Baishakhi; Kumar, Gopinatha Suresh

2014-10-01

A comprehensive study on the binding of phenazinium dyes viz. janus green B, indoine blue, safranine O and phenosafranine with double stranded poly(A) using various spectroscopic and calorimetric techniques is presented. A higher binding of janus green B and indoine blue over safranine O and phenosafranine to poly(A) was observed from all experiments. Intercalative mode of binding of the dyes was inferred from fluorescence polarization anisotropy, iodide quenching and viscosity experiments. Circular dichroism study revealed significant perturbation of the secondary structure of poly(A) on binding of these dyes. Results from isothermal titration calorimetry experiments suggested that the binding was predominantly entropy driven with a minor contribution of enthalpy to the standard molar Gibbs energy. The results presented here may open new opportunities in the application of these dyes as RNA targeted therapeutic agents.

Great interactions: How binding incorrect partners can teach us about protein recognition and function.

PubMed

Vamparys, Lydie; Laurent, Benoist; Carbone, Alessandra; Sacquin-Mora, Sophie

2016-10-01

Protein-protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross-docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross-docking predictions using the area under the specificity-sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding-site predictions resulting from the cross-docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408-1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Characterization of the high affinity binding of epsilon toxin from Clostridium perfringens to the renal system.

PubMed

Dorca-Arévalo, Jonatan; Martín-Satué, Mireia; Blasi, Juan

2012-05-25

Epsilon toxin (ε-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxaemia in livestock. In the renal system, the toxin binds to target cells before oligomerization, pore formation and cell death. Still, there is little information about the cellular and molecular mechanism involved in the initial steps of the cytotoxic action of ε-toxin, including the specific binding to the target sensitive cells. In the present report, the binding step of ε-toxin to the MDCK cell line is characterized by means of an ELISA-based binding assay with recombinant ε-toxin-green fluorescence protein (ε-toxin-GFP) and ε-prototoxin-GFP. In addition, different treatments with Pronase E, detergents, N-glycosidase F and beta-elimination on MDCK cells and renal cryosections have been performed to further characterize the ε-toxin binding. The ELISA assays revealed a single binding site with a similar dissociation constant (K(d)) for ε-toxin-GFP and ε-prototoxin-GFP, but a three-fold increase in B(max) levels in the case of ε-toxin-GFP. Double staining on kidney cryoslices with lectins and ε-prototoxin-GFP revealed specific binding to distal and collecting tubule cells. In addition, experiments on kidney and bladder cryoslices demonstrated the specific binding to distal tubule of a range of mammalian renal systems. Pronase E and beta-elimination treatments on kidney cryoslices and MDCK cells revealed that the binding of ε-toxin in renal system is mediated by a O-glycoprotein. Detergent treatments revealed that the integrity of the plasma membrane is required for the binding of ε-toxin to its receptor. Copyright © 2011 Elsevier B.V. All rights reserved.

Multi-task learning for cross-platform siRNA efficacy prediction: an in-silico study

PubMed Central

2010-01-01

Background Gene silencing using exogenous small interfering RNAs (siRNAs) is now a widespread molecular tool for gene functional study and new-drug target identification. The key mechanism in this technique is to design efficient siRNAs that incorporated into the RNA-induced silencing complexes (RISC) to bind and interact with the mRNA targets to repress their translations to proteins. Although considerable progress has been made in the computational analysis of siRNA binding efficacy, few joint analysis of different RNAi experiments conducted under different experimental scenarios has been done in research so far, while the joint analysis is an important issue in cross-platform siRNA efficacy prediction. A collective analysis of RNAi mechanisms for different datasets and experimental conditions can often provide new clues on the design of potent siRNAs. Results An elegant multi-task learning paradigm for cross-platform siRNA efficacy prediction is proposed. Experimental studies were performed on a large dataset of siRNA sequences which encompass several RNAi experiments recently conducted by different research groups. By using our multi-task learning method, the synergy among different experiments is exploited and an efficient multi-task predictor for siRNA efficacy prediction is obtained. The 19 most popular biological features for siRNA according to their jointly importance in multi-task learning were ranked. Furthermore, the hypothesis is validated out that the siRNA binding efficacy on different messenger RNAs(mRNAs) have different conditional distribution, thus the multi-task learning can be conducted by viewing tasks at an "mRNA"-level rather than at the "experiment"-level. Such distribution diversity derived from siRNAs bound to different mRNAs help indicate that the properties of target mRNA have important implications on the siRNA binding efficacy. Conclusions The knowledge gained from our study provides useful insights on how to analyze various cross-platform RNAi data for uncovering of their complex mechanism. PMID:20380733

Multi-task learning for cross-platform siRNA efficacy prediction: an in-silico study.

PubMed

Liu, Qi; Xu, Qian; Zheng, Vincent W; Xue, Hong; Cao, Zhiwei; Yang, Qiang

2010-04-10

Gene silencing using exogenous small interfering RNAs (siRNAs) is now a widespread molecular tool for gene functional study and new-drug target identification. The key mechanism in this technique is to design efficient siRNAs that incorporated into the RNA-induced silencing complexes (RISC) to bind and interact with the mRNA targets to repress their translations to proteins. Although considerable progress has been made in the computational analysis of siRNA binding efficacy, few joint analysis of different RNAi experiments conducted under different experimental scenarios has been done in research so far, while the joint analysis is an important issue in cross-platform siRNA efficacy prediction. A collective analysis of RNAi mechanisms for different datasets and experimental conditions can often provide new clues on the design of potent siRNAs. An elegant multi-task learning paradigm for cross-platform siRNA efficacy prediction is proposed. Experimental studies were performed on a large dataset of siRNA sequences which encompass several RNAi experiments recently conducted by different research groups. By using our multi-task learning method, the synergy among different experiments is exploited and an efficient multi-task predictor for siRNA efficacy prediction is obtained. The 19 most popular biological features for siRNA according to their jointly importance in multi-task learning were ranked. Furthermore, the hypothesis is validated out that the siRNA binding efficacy on different messenger RNAs(mRNAs) have different conditional distribution, thus the multi-task learning can be conducted by viewing tasks at an "mRNA"-level rather than at the "experiment"-level. Such distribution diversity derived from siRNAs bound to different mRNAs help indicate that the properties of target mRNA have important implications on the siRNA binding efficacy. The knowledge gained from our study provides useful insights on how to analyze various cross-platform RNAi data for uncovering of their complex mechanism.

Nature of the Active Sites for CO Reduction on Copper Nanoparticles; Suggestions for Optimizing Performance.

PubMed

Cheng, Tao; Xiao, Hai; Goddard, William A

2017-08-30

Recent experiments show that the grain boundaries (GBs) of copper nanoparticles (NPs) lead to an outstanding performance in reducing CO 2 and CO to alcohol products. We report here multiscale simulations that simulate experimental synthesis conditions to predict the structure of a 10 nm Cu NP (158 555 atoms). To identify active sites, we first predict the CO binding at a large number of sites and select four exhibiting CO binding stronger than the (211) step surface. Then, we predict the formation energy of the *OCCOH intermediate as a descriptor for C-C coupling, identifying two active sites, both of which have an under-coordinated surface square site adjacent to a subsurface stacking fault. We then propose a periodic Cu surface (4 by 4 supercell) with a similar site that substantially decreases the formation energy of *OCCOH, by 0.14 eV.

The discovery of [Ni(NHC)RCN]2 species and their role as cycloaddition catalysts for the formation of pyridines.

PubMed

Stolley, Ryan M; Duong, Hung A; Thomas, David R; Louie, Janis

2012-09-12

The reaction of Ni(COD)(2), IPr, and nitrile affords dimeric [Ni(IPr)RCN](2) in high yields. X-ray analysis revealed these species display simultaneous η(1)- and η(2)-nitrile binding modes. These dimers are catalytically competent in the formation of pyridines from the cycloaddition of diynes and nitriles. Kinetic analysis showed the reaction to be first order in [Ni(IPr)RCN](2), zeroth order in added IPr, zeroth order in nitrile, and zeroth order in diyne. Extensive stoichiometric competition studies were performed, and selective incorporation of the exogenous, not dimer bound, nitrile was observed. Post cycloaddition, the dimeric state was found to be largely preserved. Nitrile and ligand exchange experiments were performed and found to be inoperative in the catalytic cycle. These observations suggest a mechanism whereby the catalyst is activated by partial dimer-opening followed by binding of exogenous nitrile and subsequent oxidative heterocoupling.

The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1.

PubMed Central

Toczyski, D P; Steitz, J A

1993-01-01

EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function. Images PMID:8380232

A versatile assay for RNA-binding proteins in living cells

PubMed Central

Strein, Claudia; Alleaume, Anne-Marie; Rothbauer, Ulrich; Hentze, Matthias W.; Castello, Alfredo

2014-01-01

RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein–mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology. PMID:24664470

Using FRET to Measure the Angle at Which a Protein Bends DNA: TBP Binding a TATA Box as a Model System

ERIC Educational Resources Information Center

Kugel, Jennifer F.

2008-01-01

An undergraduate biochemistry laboratory experiment that will teach the technique of fluorescence resonance energy transfer (FRET) while analyzing protein-induced DNA bending is described. The experiment uses the protein TATA binding protein (TBP), which is a general transcription factor that recognizes and binds specific DNA sequences known as…

Dissociation of binding and learning processes.

PubMed

Moeller, Birte; Frings, Christian

2017-11-01

A single encounter of a stimulus together with a response can result in a short-lived association between the stimulus and the response [sometimes called an event file, see Hommel, Müsseler, Aschersleben, & Prinz, (2001) Behavioral and Brain Sciences, 24, 910-926]. The repetition of stimulus-response pairings typically results in longer lasting learning effects indicating stimulus-response associations (e.g., Logan & Etherton, (1994) Journal of Experimental Psychology: Learning, Memory, and Cognition, 20, 1022-1050]. An important question is whether or not what has been described as stimulus-response binding in action control research is actually identical with an early stage of incidental learning (e.g., binding might be seen as single-trial learning). Here, we present evidence that short-lived binding effects can be distinguished from learning of longer lasting stimulus-response associations. In two experiments, participants always responded to centrally presented target letters that were flanked by response irrelevant distractor letters. Experiment 1 varied whether distractors flanked targets on the horizontal or vertical axis. Binding effects were larger for a horizontal than for a vertical distractor-target configuration, while stimulus configuration did not influence incidental learning of longer lasting stimulus-response associations. In Experiment 2, the duration of the interval between response n - 1 and presentation of display n (500 ms vs. 2000 ms) had opposing influences on binding and learning effects. Both experiments indicate that modulating factors influence stimulus-response binding and incidental learning effects in different ways. We conclude that distinct underlying processes should be assumed for binding and incidental learning effects.

Anchoring protein crystals to mounting loops with hydrogel using inkjet technology.

PubMed

Shinoda, Akira; Tanaka, Yoshikazu; Yao, Min; Tanaka, Isao

2014-11-01

X-ray crystallography is an important technique for structure-based drug discovery, mainly because it is the only technique that can reveal whether a ligand binds to the target protein as well as where and how it binds. However, ligand screening by X-ray crystallography involves a crystal-soaking experiment, which is usually performed manually. Thus, the throughput is not satisfactory for screening large numbers of candidate ligands. In this study, a technique to anchor protein crystals to mounting loops by using gel and inkjet technology has been developed; the method allows soaking of the mounted crystals in ligand-containing solution. This new technique may assist in the design of a fully automated drug-screening pipeline.

Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

PubMed

Raghav, Sunil Kumar; Deplancke, Bart

2012-01-01

Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

Quantitative modeling of peptide binding to TAP using support vector machine.

PubMed

Diez-Rivero, Carmen M; Chenlo, Bernardo; Zuluaga, Pilar; Reche, Pedro A

2010-01-01

The transport of peptides to the endoplasmic reticulum by the transporter associated with antigen processing (TAP) is a necessary step towards determining CD8 T cell epitopes. In this work, we have studied the predictive performance of support vector machine models trained on single residue positions and residue combinations drawn from a large dataset consisting of 613 nonamer peptides of known affinity to TAP. Predictive performance of these TAP affinity models was evaluated under 10-fold cross-validation experiments and measured using Pearson's correlation coefficients (R(p)). Our results show that every peptide position (P1-P9) contributes to TAP binding (minimum R(p) of 0.26 +/- 0.11 was achieved by a model trained on the P6 residue), although the largest contributions to binding correspond to the C-terminal end (R(p) = 0.68 +/- 0.06) and the P1 (R(p) = 0.51 +/- 0.09) and P2 (0.57 +/- 0.08) residues of the peptide. Training the models on additional peptide residues generally improved their predictive performance and a maximum correlation (R(p) = 0.89 +/- 0.03) was achieved by a model trained on the full-length sequences or a residue selection consisting of the first 5 N- and last 3 C-terminal residues of the peptides included in the training set. A system for predicting the binding affinity of peptides to TAP using the methods described here is readily available for free public use at http://imed.med.ucm.es/Tools/tapreg/. (c) 2009 Wiley-Liss, Inc.

What Do We Learn from Binding Features? Evidence for Multilevel Feature Integration

ERIC Educational Resources Information Center

Colzato, Lorenza S.; Raffone, Antonino; Hommel, Bernhard

2006-01-01

Four experiments were conducted to investigate the relationship between the binding of visual features (as measured by their after-effects on subsequent binding) and the learning of feature-conjunction probabilities. Both binding and learning effects were obtained, but they did not interact. Interestingly, (shape-color) binding effects…

Kir6.2-dependent high-affinity repaglinide binding to β-cell KATP channels

PubMed Central

Hansen, Ann Maria K; Hansen, John Bondo; Carr, Richard D; Ashcroft, Frances M; Wahl, Philip

2005-01-01

The β-cell KATP channel is composed of two types of subunit – the inward rectifier K+ channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N-terminus (Kir6.2ΔN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2ΔN14/SUR1 channels. All binding experiments are performed on membranes in ATP-free buffer at 37°C. Repaglinide was found to bind with low affinity (KD=59±16 nM) to SUR1 alone, but with high affinity (increased ∼150-fold) when SUR1 was co-expressed with Kir6.2 (KD=0.42±0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. Repaglinide bound with low affinity (KD=51±23 nM) to SUR1 co-expressed with Kir6.2ΔN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild-type channels. In whole-cell patch-clamp experiments inhibition of Kir6.2ΔN14/SUR1 currents by both repaglinide and nateglinde is abolished. The results suggest that Kir6.2 causes a conformational change in SUR1 required for high-affinity repaglinide binding, or that the high-affinity repaglinide-binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1. PMID:15678092

Chemical modification of protein A chromatography ligands with polyethylene glycol. I: Effects on IgG adsorption equilibrium, kinetics, and transport.

PubMed

Weinberg, Justin; Zhang, Shaojie; Crews, Gillian; Carta, Giorgio; Przybycien, Todd

2018-04-20

Chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) has been proposed as a strategy to increase the process selectivity and resin robustness by providing the ligand with a steric repulsion barrier against non-specific binding. This article comprises a comprehensive study of IgG adsorption and transport in Repligen CaptivA PriMAB resin with PEGylated ProA ligands that are modified using 5.2 and 21.5 kDa PEG chains. We studied the impact of the molecular weight of the PEG as well as the extent of PEGylation for the 5.2 kDa PEG modification. In all cases, PEGylation of ProA ligands decreases the resin average pore size, particle porosity, and static binding capacity for IgG proportional to the volume of conjugated PEG in the resin. Resin batch uptake experiments conducted in bulk via a stirred-tank system and with individual resin particles under confocal laser scanning microscopy suggests that PEGylation introduces heterogeneity into IgG binding kinetics: a fraction of the IgG binding sites are transformed from typical fast association kinetic behavior to slow kinetic behavior. pH gradient elution experiments of an IgG molecule on the modified resins show an increase in IgG elution pH for all modified resins, implying a decrease in IgG-ProA binding affinity on modification. Despite losses in static binding capacity for all resins with PEGylated ligands, the loss of dynamic binding capacity at 10% breakthrough (DBC 10% ) ranged more broadly from almost 0-47% depending on the PEG molecular weight and the extent of PEGylation. Minimal losses in DBC 10% were observed with a low extent of PEGylation with a smaller molecular weight PEG, while higher losses were observed at higher extents of PEGylation and with higher molecular weight PEG due to decreased static binding capacity and increased mass transfer resistance. This work provides insight into the practical implications for resin performance if PEGylation is considered as a strategy for selectivity enhancement in affinity chromatography with macromolecular ligands. Copyright © 2018 Elsevier B.V. All rights reserved.

A high pressure study of calmodulin-ligand interactions using small-angle X-ray and elastic incoherent neutron scattering.

PubMed

Cinar, Süleyman; Al-Ayoubi, Samy; Sternemann, Christian; Peters, Judith; Winter, Roland; Czeslik, Claus

2018-01-31

Calmodulin (CaM) is a Ca 2+ sensor and mediates Ca 2+ signaling through binding of numerous target ligands. The binding of ligands by Ca 2+ -saturated CaM (holo-CaM) is governed by attractive hydrophobic and electrostatic interactions that are weakened under high pressure in aqueous solutions. Moreover, the potential formation of void volumes upon ligand binding creates a further source of pressure sensitivity. Hence, high pressure is a suitable thermodynamic variable to probe protein-ligand interactions. In this study, we compare the binding of two different ligands to holo-CaM as a function of pressure by using X-ray and neutron scattering techniques. The two ligands are the farnesylated hypervariable region (HVR) of the K-Ras4B protein, which is a natural binding partner of holo-CaM, and the antagonist trifluoperazine (TFP), which is known to inhibit holo-CaM activity. From small-angle X-ray scattering experiments performed up to 3000 bar, we observe a pressure-induced partial unfolding of the free holo-CaM in the absence of ligands, where the two lobes of the dumbbell-shaped protein are slightly swelled. In contrast, upon binding TFP, holo-CaM forms a closed globular conformation, which is pressure stable at least up to 3000 bar. The HVR of K-Ras4B shows a different binding behavior, and the data suggest the dissociation of the holo-CaM/HVR complex under high pressure, probably due to a less dense protein contact of the HVR as compared to TFP. The elastic incoherent neutron scattering experiments corroborate these findings. Below 2000 bar, pressure induces enhanced atomic fluctuations in both holo-CaM/ligand complexes, but those of the holo-CaM/HVR complex seem to be larger. Thus, the inhibition of holo-CaM by TFP is supported by a low-volume ligand binding, albeit this is not associated with a rigidification of the complex structure on the sub-ns Å-scale.

pH and Protein Sensing with Functionalized Semiconducting Oxide Nanobelt FETs

NASA Astrophysics Data System (ADS)

Cheng, Yi; Yun, C. S.; Strouse, G. F.; Xiong, P.; Yang, R. S.; Wang, Z. L.

2008-03-01

We report solution pH sensing and selective protein detection with high-performance channel-limited field-effect transistors (FETs) based on single semiconducting oxide (ZnO and SnO2) nanobelts^1. The devices were integrated with PDMS microfluidic channels for analyte delivery and the source/drain contacts were passivated for in-solution sensing. pH sensing experiments were performed on FETs with functionalized and unmodified nanobelts. Functionalization of the nanobelts by APTES was found to greatly improve the pH sensitivity. The change in nanobelt conductance as functions of pH values at different gate voltages and ionic strengths showed high sensitivity and consistency. For the protein detection, we achieved highly selective biotinylation of the nanobelt channel with through APTES linkage. The specific binding of fluorescently-tagged streptavidin to the biotinylated nanobelt was verified by fluorescence microscopy; non-specific binding to the substrate was largely eliminated using PEG-silane passivation. The electrical responses of the biotinylated FETs to the streptavidin binding in PBS buffers of different pH values were systematically measured. The results will be presented and discussed. ^1Y. Cheng et al., Appl. Phys. Lett. 89, 093114 (2006). *Supported by NSF NIRT Grant ECS-0210332.

Integration Method of Emphatic Motions and Adverbial Expressions with Scalar Parameters for Robotic Motion Coaching System

NASA Astrophysics Data System (ADS)

Okuno, Keisuke; Inamura, Tetsunari

A robotic coaching system can improve humans' learning performance of motions by intelligent usage of emphatic motions and adverbial expressions according to user reactions. In robotics, however, method to control both the motions and the expressions and how to bind them had not been adequately discussed from an engineering point of view. In this paper, we propose a method for controlling and binding emphatic motions and adverbial expressions by using two scalar parameters in a phase space. In the phase space, variety of motion patterns and verbal expressions are connected and can be expressed as static points. We show the feasibility of the proposing method through experiments of actual sport coaching tasks for beginners. From the results of participants' improvements in motion learning, we confirmed the feasibility of the methods to control and bind emphatic motions and adverbial expressions, as well as confirmed contribution of the emphatic motions and positive correlation of adverbial expressions for participants' improvements in motion learning. Based on the results, we introduce a hypothesis that individually optimized method for binding adverbial expression is required.

What Controls the Limit of Supercooling and Superheating of Pinned Ice Surfaces?

PubMed

Naullage, Pavithra M; Qiu, Yuqing; Molinero, Valeria

2018-04-05

Cold-adapted organisms produce antifreeze proteins and glycoproteins to control the growth, melting and recrystallization of ice. It has been proposed that these molecules pin the crystal surface, creating a curvature that arrests the growth and melting of the crystal. Here we use thermodynamic modeling and molecular simulations to demonstrate that the curvature of the superheated or supercooled surface depends on the temperature and distances between ice-binding molecules, but not the details of their interactions with ice. We perform simulations of ice pinned with the antifreeze protein TmAFP, polyvinyl alcohol with different degrees of polymerization, and model ice-binding molecules to determine the thermal hystereses on melting and freezing, i.e. the maximum curvature that can be attained before, respectively, ice melts or grows irreversibly over the ice-binding molecules. We find that the thermal hysteresis is controlled by the bulkiness of the ice-binding molecules and their footprint at the ice surface. We elucidate the origin of the asymmetry between freezing and melting hysteresis found in experiments and propose guidelines to design synthetic antifreeze molecules with potent thermal hysteresis activity.

Determination of layer-dependent exciton binding energies in few-layer black phosphorus

PubMed Central

Zhang, Guowei; Chaves, Andrey; Huang, Shenyang; Wang, Fanjie; Xing, Qiaoxia; Low, Tony; Yan, Hugen

2018-01-01

The attraction between electrons and holes in semiconductors forms excitons, which largely determine the optical properties of the hosting material, and hence the device performance, especially for low-dimensional systems. Mono- and few-layer black phosphorus (BP) are emerging two-dimensional (2D) semiconductors. Despite its fundamental importance and technological interest, experimental investigation of exciton physics has been rather limited. We report the first systematic measurement of exciton binding energies in ultrahigh-quality few-layer BP by infrared absorption spectroscopy, with layer (L) thickness ranging from 2 to 6 layers. Our experiments allow us to determine the exciton binding energy, decreasing from 213 meV (2L) to 106 meV (6L). The scaling behavior with layer numbers can be well described by an analytical model, which takes into account the nonlocal screening effect. Extrapolation to free-standing monolayer yields a large binding energy of ~800 meV. Our study provides insights into 2D excitons and their crossover from 2D to 3D, and demonstrates that few-layer BP is a promising high-quality optoelectronic material for potential infrared applications. PMID:29556530

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

PubMed Central

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

PubMed

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

PubMed

Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

2017-12-01

Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA

NASA Astrophysics Data System (ADS)

Afrin, Shumaila; Rahman, Yusra; Sarwar, Tarique; Husain, Mohammed Amir; Ali, Abad; Shamsuzzaman; Tabish, Mohammad

2017-11-01

Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV-visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103 M- 1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.

Theory of long binding events in single-molecule–controlled rotation experiments on F1-ATPase

PubMed Central

Volkán-Kacsó, Sándor; Marcus, Rudolph A.

2017-01-01

The theory of elastic group transfer for the binding and release rate constants for nucleotides in F1-ATPase as a function of the rotor angle is further extended in several respects. (i) A method is described for predicting the experimentally observed lifetime distribution of long binding events in the controlled rotation experiments by taking into account the hydrolysis and synthesis reactions occurring during these events. (ii) A method is also given for treating the long binding events in the experiments and obtaining the rate constants for the hydrolysis and synthesis reactions occurring during these events. (iii) The theory in the previous paper is given in a symmetric form, an extension that simplifies the application of the theory to experiments. It also includes a theory-based correction of the reported “on” and “off” rates by calculating the missed events. A near symmetry of the data about the angle of −40° and a “turnover” in the binding rate data vs. rotor angle for angles greater than ∼40° is also discussed. PMID:28652332

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

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

Wada, Takeyoshi; Asahi, Toru; Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480

2016-08-26

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as amore » thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.« less

Preclinical evaluation of a bispecific low-molecular heterodimer targeting both PSMA and GRPR for improved PET imaging and therapy of prostate cancer.

PubMed

Eder, Matthias; Schäfer, Martin; Bauder-Wüst, Ulrike; Haberkorn, Uwe; Eisenhut, Michael; Kopka, Klaus

2014-05-01

It has recently been reported that metastases of prostate cancer usually show highly heterogeneous or partly lost prostate-specific membrane antigen (PSMA) expression. In order to image and treat both PSMA positive and negative tissues PSMA targeting probes need to be extended by a further specificity. Since prostate cancer cells usually express both PSMA and gastrin-releasing peptide receptor (GRPR) a bispecific low-molecular heterodimeric molecule, addressing both targets at the same time, may significantly improve prostate cancer imaging and therapy. The nonapeptide BZH3 representing the GRPR binding part was combined with the urea-based PSMA inhibitor Glu-urea-Lys(Ahx)-HBED-CC. The syntheses of the compounds were performed according to standard Fmoc-solid phase peptide synthesis. The binding properties were analyzed by competitive cell binding and internalization experiments. The in vivo targeting properties were investigated by means of biodistribution studies. Cell binding experiments revealed high binding affinities to both GRPR and PSMA expressing cell lines. The heterodimer bound with IC50 -values essentially matching the IC50 values of the respective monomers (25.0 ± 5.4 nM for PSMA and 9.0 ± 1.8 nM for GRPR, respectively). In vivo, the heterodimer showed dual targeting of PSMA (5.4%ID/g for PSMA-positive tumors) and GRPR receptors (3.3% ID/g for GRPR-positive tumors) while exhibiting fast pharmacokinetic properties. The clearance from background was comparable to the monomeric PSMA-targeting reference. The heterodimeric molecule is a promising agent for PET imaging of primary and recurrent prostate cancer covering two receptor entities which might lead to an improved diagnostic sensitivity and therapeutic efficiency. © 2014 Wiley Periodicals, Inc.

99mTc-HYNIC-(tricine/EDDA)-FROP peptide for MCF-7 breast tumor targeting and imaging.

PubMed

Ahmadpour, Sajjad; Noaparast, Zohreh; Abedi, Seyed Mohammad; Hosseinimehr, Seyed Jalal

2018-02-19

Breast cancer is the most common malignancy among women in the world. Development of novel tumor-specific radiopharmaceuticals for early breast tumor diagnosis is highly desirable. In this study we developed 99m Tc-HYNIC-(tricine/EDDA)-Lys-FROP peptide with the ability of specific binding to MCF-7 breast tumor. The FROP-1 peptide was conjugated with the bifunctional chelator hydrazinonicotinamide (HYNIC) and labeled with 99m Tc using tricine/EDDA co-ligand. The cellular specific binding of 99m Tc-HYNIC-FROP was evaluated on different cell lines as well as with blocking experiment on MCF-7 (human breast adenocarcinoma). The tumor targeting and imaging of this labeled peptide were performed on MCF-7 tumor bearing mice. Radiochemical purity for 99m Tc-HYNIC-(tricine/EDDA)-FROP was 99% which was determined with ITLC method. This radiolabeled peptide showed high stability in normal saline and serum about 98% which was monitored with HPLC method. In saturation binding experiments, the binding constant (K d ) to MCF-7 cells was determined to be 158 nM. Biodistribution results revealed that the 99m Tc-HYNIC-FROP was mainly exerted from urinary route. The maximum tumor uptake was found after 30 min post injection (p.i.); however maximum tumor/muscle ratio was seen at 15 min p.i. The tumor uptake of this labeled peptide was specific and blocked by co-injection of excess FROP. According to the planar gamma imaging result, tumor was clearly visible due to the tumor uptake of 99m Tc-HYNIC-(tricine/EDDA)-FROP in mouse after 15 min p.i. The 99m Tc-HYNIC-(tricine/EDDA)-FROP is considered a promising probe with high specific binding to MCF-7 breast cancer cells.

Role of Microbial Exopolymeric Substances (EPS) on Chromium Sorption and Transport in Heterogeneous Subsurface Soils: I. Cr(III) Complexation with EPS in Aqueous Solution

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

C Kantar; H Demiray; N Dogan

2011-12-31

Chromium (III) binding by exopolymeric substances (EPS) isolated from Pseudomonas putida P18, Pseudomonas aeruginosa P16 and Pseudomonas stutzeri P40 strains were investigated by the determination of conditional stability constants and the concentration of functional groups using the ion-exchange experiments and potentiometric titrations. Spectroscopic (EXAFS) analysis was also used to obtain information on the nature of Cr(III) binding with EPS functional groups. The data from ion-exchange experiments and potentiometric titrations were evaluated using a non-electrostatic discrete ligand approach. The modeling results show that the acid/base properties of EPSs can be best characterized by invoking four different types of acid functional groupsmore » with arbitrarily assigned pK{sub a} values of 4, 6, 8 and 10. The analysis of ion-exchange data using the discrete ligand approach suggests that while the Cr binding by EPS from P. aeruginosa can be successfully described based on a reaction stoichiometry of 1:2 between Cr(III) and HL{sub 2} monoprotic ligands, the accurate description of Cr binding by EPSs extracted from P. putida and P. stutzeri requires postulation of 1:1 Cr(III)-ligand complexes with HL{sub 2} and HL{sub 3} monoprotic ligands, respectively. These results indicate that the carboxyl and/or phosphoric acid sites contribute to Cr(III) binding by microbial EPS, as also confirmed by EXAFS analysis performed in the current study. Overall, this study highlights the need for incorporation of Cr-EPS interactions into transport and speciation models to more accurately assess microbial Cr(VI) reduction and chromium transport in subsurface systems, including microbial reactive treatment barriers.« less

Role of microbial exopolymeric substances (EPS) on chromium sorption and transport in heterogeneous subsurface soils: I. Cr(III) complexation with EPS in aqueous solution

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

Kantar, C.; Dodge, C.; Demiray, H.

2011-01-26

Chromium (III) binding by exopolymeric substances (EPS) isolated from Pseudomonas putida P18, Pseudomonas aeruginosa P16 and Pseudomonas stutzeri P40 strains were investigated by the determination of conditional stability constants and the concentration of functional groups using the ion-exchange experiments and potentiometric titrations. Spectroscopic (EXAFS) analysis was also used to obtain information on the nature of Cr(III) binding with EPS functional groups. The data from ion-exchange experiments and potentiometric titrations were evaluated using a non-electrostatic discrete ligand approach. The modeling results show that the acid/base properties of EPSs can be best characterized by invoking four different types of acid functional groupsmore » with arbitrarily assigned pK{sub a} values of 4, 6, 8 and 10. The analysis of ion-exchange data using the discrete ligand approach suggests that while the Cr binding by EPS from P. aeruginosa can be successfully described based on a reaction stoichiometry of 1:2 between Cr(III) and HL{sub 2} monoprotic ligands, the accurate description of Cr binding by EPSs extracted from P. putida and P. stutzeri requires postulation of 1:1 Cr(III)-ligand complexes with HL{sub 2} and HL{sub 3} monoprotic ligands, respectively. These results indicate that the carboxyl and/or phosphoric acid sites contribute to Cr(III) binding by microbial EPS, as also confirmed by EXAFS analysis performed in the current study. Overall, this study highlights the need for incorporation of Cr-EPS interactions into transport and speciation models to more accurately assess microbial Cr(VI) reduction and chromium transport in subsurface systems, including microbial reactive treatment barriers.« less

Conformational selection in protein binding and function

PubMed Central

Weikl, Thomas R; Paul, Fabian

2014-01-01

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

Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis.

PubMed

Mozafari, Mona; El Deeb, Sami; Krull, Friederike; Wildgruber, Robert; Weber, Gerhard; Reiter, Christian G; Wätzig, Hermann

2018-02-01

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/R f ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

PubMed Central

Chen, Qiang; Cheng, Mary Hongying; Xu, Yan; Tang, Pei

2010-01-01

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (KD) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins. PMID:20858424

Neuroscience: toward unbinding the binding problem.

PubMed

Whitney, David

2009-03-24

How the brain 'binds' information to create a coherent perceptual experience is an enduring question. Recent research in the psychophysics of perceptual binding and developments in fMRI analysis techniques are bringing us closer to an understanding of how the brain solves the binding problem.

Spectroscopic research of Lambda hypernuclei up to medium-heavy mass region with the (e,e'K +) reaction

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

Gogami, Toshiyuki

In 2009 (August-November), the E05-115 experiment was carried out at JLab to investigate L hypernuclei in the wide mass region up to A = 52 (more » $$7\\atop{Λ}$$He, $$10\\atop{Λ}$$Be, $$12\\atop{Λ}$$B and $$52\\atop{Λ}$$V) with the (e,e'K +) reaction. This is the first attempt to investigate a medium heavy L hypernucleus with the (e,e'K +) reaction. Experimentally, it is difficult to measure heavier L hypernuclei as background rates of particles which originate from electromagnetic processes are roughly in proportion to Z2 (Z: target proton number) in the (e,e'K +) experiment. To perform the experiment, many experimental techniques have been developed and introduced such as optimization of the electron spectrometer configuration (tilt method), clean kaon identification, particle tracking under high multiplicity environment, precise energy scale calibration and so on. In the present thesis, experimental results of the elementary process of p(e,e'K +)L, L hypernuclei of $$7\\atop{Λ}$$He, $$10\\atop{Λ}$$Be, $$12\\atop{Λ}$$B and $$52\\atop{Λ}$$V are shown. Elementary processes of the electroproduction of L and Σ 0, p(e,e'K +)L, Σ 0 were used for the absolute energy scale calibration of our spectrometer systems. A careful Monte Carlo simulation shows that the binding energy can be obtained with a systematic error of 0.11 MeV with our energy scale calibration method. A study of the elementary process of L is important to understand L hypernuclei as it is essential for theoretical calculations of L hypernuclei. The differential cross section of the p(e,e'K +)L reaction at the small K + scattering angle (theta-CM/gamma-K approx. 15.5°), the small Q 2 (approx 0.01 [GeV/c] 2) and the total energy of W = 1.92 GeV, where no experimental data exists was obtained to be 235 ± 13$$+28\\atop{-24}$$ nb/sr. The ground state (1/2 +) binding energy of $$7\\atop{Λ}$$He was already measured in JLab E01-011 (2005). In the present work, the binding energy of 1/2 + state was determined to be B Λ = 5.55 ± 0.10 ±0.11 MeV with five times more statistic and smaller systematic errors than those of the previous experiment. The ground state binding energy is important to test the phe- nomenologically introduced CSB (Charge Symmetry Breaking) LN interaction for A = 7, T = 1 hypernuclear systems. In addition, a peak which is interpreted as 3/2 + and 5/2 + states was measured to be B Λ = 3.65 ± 0.20 ±0.11 MeV with sufficient statistic for the first time. Only three events of the ground state of $$10\\atop{Λ}$$ Be had been observed in the emulsion experiments. The present experiment is the first spectroscopic measurement of 10/L-Be, and the detailed structures have been successfully measured for the first time. About three times better energy resolution was achieved in the present experiment (0.78 MeV in FWHM) than that of the mirror L hy- pernucleus, $$10\\atop{Λ}$$B (2.2 MeV in FWHM) which was measured in the (π +,K +) experiment at KEK. The result of the ground state binding energy was obtained to be B Λ = 8.55 ± 0.07 ± 0.11 MeV which serves also to discuss about the CSB effect in the LN interaction. $$12\\atop{Λ}$$B has been measured with the world best energy resolution of 0:5 MeV (FWHM) among the reaction spectroscopy of L hypernuclei. The results of $$12\\atop{Λ}$$B are compared with the experimental results in the previous experiments to confirm the consistency. Furthermore, the obtained ground state binding energies of $$12\\atop{Λ}$$B (B Λ= 11.38 ± 0.02 ± 0.11 MeV) and $$52\\atop{Λ}$$V (B Λ = 21.88 ± 0.59 ± 0.11 MeV) indicate that the reported value of 12/L-C which has been used as a reference of binding energy measurements for the (π +,K +) experiments would be shallower by ~ 0.5 MeV. A pilot study for investigation in the medium-heavy mass region with the (e,e'K +) experiment was performed by measuring $$52\\atop{Λ}$$V. The ground state binding energy of $$52\\atop{Λ}$$V has been measured, overcoming high multiplicity environment. The results are discussed with the ex- perimental results of 51/L-V measured at KEK. The present result is the first measurement of L's binding energy of the ground state without the emulsion reference in the medium-heavy mass region, which could be a substantial improvement in the information needed for understanding the single particle potential of L. In the present experiment, L hypernuclear measurement with a small systematic error of ~ 0.1 MeV by the (e,e'K +) reaction has been established. Moreover, the present work opened a door to the heavier L hypernuclear measurement with the (e,e'K +) reaction in the future.« less

The Binding Constant of Estradiol to Bovine Serum Albumin: An Upper-Level Experiment Utilizing Tritium-Labeled Estradiol and Liquid Scintillation Counting

ERIC Educational Resources Information Center

Peihong Liang; Adhyaru, Bhavin; Pearson, Wright L.; Williams, Kathryn R.

2006-01-01

The experiment used [to the third power]H-labeled estradiol to determine the binding constant of estradiol to bovine serum albumin. Estradiol must complex with serum proteins for the transport in the blood stream because of its low solubility in aqueous systems and estradiol-protein binding constant, where K[subscript B] is important to understand…

Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

PubMed

Mozafari, Mona; Balasupramaniam, Shantheya; Preu, Lutz; El Deeb, Sami; Reiter, Christian G; Wätzig, Hermann

2017-06-01

A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/R f ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rational Design of a New Class of Toll-Like Receptor 4 (TLR4) Tryptamine Related Agonists by Means of the Structure- and Ligand-Based Virtual Screening for Vaccine Adjuvant Discovery.

PubMed

Honegr, Jan; Dolezal, Rafael; Malinak, David; Benkova, Marketa; Soukup, Ondrej; Almeida, Joyce S F D de; Franca, Tanos C C; Kuca, Kamil; Prymula, Roman

2018-01-04

In order to identify novel lead structures for human toll-like receptor 4 ( h TLR4) modulation virtual high throughput screening by a peta-flops-scale supercomputer has been performed. Based on the in silico studies, a series of 12 compounds related to tryptamine was rationally designed to retain suitable molecular geometry for interaction with the h TLR4 binding site as well as to satisfy general principles of drug-likeness. The proposed compounds were synthesized, and tested by in vitro and ex vivo experiments, which revealed that several of them are capable to stimulate h TLR4 in vitro up to 25% activity of Monophosphoryl lipid A. The specific affinity of the in vitro most potent substance was confirmed by surface plasmon resonance direct-binding experiments. Moreover, two compounds from the series show also significant ability to elicit production of interleukin 6.

Correlating single-molecule and ensemble-average measurements of peptide adsorption onto different inorganic materials.

PubMed

Kim, Seong-Oh; Jackman, Joshua A; Mochizuki, Masahito; Yoon, Bo Kyeong; Hayashi, Tomohiro; Cho, Nam-Joon

2016-06-07

The coating of solid-binding peptides (SBPs) on inorganic material surfaces holds significant potential for improved surface functionalization at nano-bio interfaces. In most related studies, the goal has been to engineer peptides with selective and high binding affinity for a target material. The role of the material substrate itself in modulating the adsorption behavior of a peptide molecule remains less explored and there are few studies that compare the interaction of one peptide with different inorganic substrates. Herein, using a combination of two experimental techniques, we investigated the adsorption of a 16 amino acid-long random coil peptide to various inorganic substrates - gold, silicon oxide, titanium oxide and aluminum oxide. Quartz crystal microbalance-dissipation (QCM-D) experiments were performed in order to measure the peptide binding affinity for inorganic solid supports at the ensemble average level, and atomic force microscopy (AFM) experiments were conducted in order to determine the adhesion force of a single peptide molecule. A positive trend was observed between the total mass uptake of attached peptide and the single-molecule adhesion force on each substrate. Peptide affinity for gold was appreciably greater than for the oxide substrates. Collectively, the results obtained in this study offer insight into the ways in which inorganic materials can differentially influence and modulate the adhesion of SBPs.

Structures of endothiapepsin-fragment complexes from crystallographic fragment screening using a novel, diverse and affordable 96-compound fragment library.

PubMed

Huschmann, Franziska U; Linnik, Janina; Sparta, Karine; Ühlein, Monika; Wang, Xiaojie; Metz, Alexander; Schiebel, Johannes; Heine, Andreas; Klebe, Gerhard; Weiss, Manfred S; Mueller, Uwe

2016-05-01

Crystallographic screening of the binding of small organic compounds (termed fragments) to proteins is increasingly important for medicinal chemistry-oriented drug discovery. To enable such experiments in a widespread manner, an affordable 96-compound library has been assembled for fragment screening in both academia and industry. The library is selected from already existing protein-ligand structures and is characterized by a broad ligand diversity, including buffer ingredients, carbohydrates, nucleotides, amino acids, peptide-like fragments and various drug-like organic compounds. When applied to the model protease endothiapepsin in a crystallographic screening experiment, a hit rate of nearly 10% was obtained. In comparison to other fragment libraries and considering that no pre-screening was performed, this hit rate is remarkably high. This demonstrates the general suitability of the selected compounds for an initial fragment-screening campaign. The library composition, experimental considerations and time requirements for a complete crystallographic fragment-screening campaign are discussed as well as the nine fully refined obtained endothiapepsin-fragment structures. While most of the fragments bind close to the catalytic centre of endothiapepsin in poses that have been observed previously, two fragments address new sites on the protein surface. ITC measurements show that the fragments bind to endothiapepsin with millimolar affinity.

Structures of endothiapepsin–fragment complexes from crystallographic fragment screening using a novel, diverse and affordable 96-compound fragment library

PubMed Central

Huschmann, Franziska U.; Linnik, Janina; Sparta, Karine; Ühlein, Monika; Wang, Xiaojie; Metz, Alexander; Schiebel, Johannes; Heine, Andreas; Klebe, Gerhard; Weiss, Manfred S.; Mueller, Uwe

2016-01-01

Crystallographic screening of the binding of small organic compounds (termed fragments) to proteins is increasingly important for medicinal chemistry-oriented drug discovery. To enable such experiments in a widespread manner, an affordable 96-compound library has been assembled for fragment screening in both academia and industry. The library is selected from already existing protein–ligand structures and is characterized by a broad ligand diversity, including buffer ingredients, carbohydrates, nucleotides, amino acids, peptide-like fragments and various drug-like organic compounds. When applied to the model protease endothiapepsin in a crystallographic screening experiment, a hit rate of nearly 10% was obtained. In comparison to other fragment libraries and considering that no pre-screening was performed, this hit rate is remarkably high. This demonstrates the general suitability of the selected compounds for an initial fragment-screening campaign. The library composition, experimental considerations and time requirements for a complete crystallographic fragment-screening campaign are discussed as well as the nine fully refined obtained endothiapepsin–fragment structures. While most of the fragments bind close to the catalytic centre of endothiapepsin in poses that have been observed previously, two fragments address new sites on the protein surface. ITC measurements show that the fragments bind to endothiapepsin with millimolar affinity. PMID:27139825

The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

PubMed Central

Campanacci, Valérie; Bishop, Russell E.; Blangy, Stéphanie; Tegoni, Mariella; Cambillau, Christian

2016-01-01

Lipocalins, a widespread multifunctional family of small proteins (15–25 kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183–188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar Kd range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar Kd range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane. PMID:16920109

DNA motif elucidation using belief propagation.

PubMed

Wong, Ka-Chun; Chan, Tak-Ming; Peng, Chengbin; Li, Yue; Zhang, Zhaolei

2013-09-01

Protein-binding microarray (PBM) is a high-throughout platform that can measure the DNA-binding preference of a protein in a comprehensive and unbiased manner. A typical PBM experiment can measure binding signal intensities of a protein to all the possible DNA k-mers (k=8∼10); such comprehensive binding affinity data usually need to be reduced and represented as motif models before they can be further analyzed and applied. Since proteins can often bind to DNA in multiple modes, one of the major challenges is to decompose the comprehensive affinity data into multimodal motif representations. Here, we describe a new algorithm that uses Hidden Markov Models (HMMs) and can derive precise and multimodal motifs using belief propagations. We describe an HMM-based approach using belief propagations (kmerHMM), which accepts and preprocesses PBM probe raw data into median-binding intensities of individual k-mers. The k-mers are ranked and aligned for training an HMM as the underlying motif representation. Multiple motifs are then extracted from the HMM using belief propagations. Comparisons of kmerHMM with other leading methods on several data sets demonstrated its effectiveness and uniqueness. Especially, it achieved the best performance on more than half of the data sets. In addition, the multiple binding modes derived by kmerHMM are biologically meaningful and will be useful in interpreting other genome-wide data such as those generated from ChIP-seq. The executables and source codes are available at the authors' websites: e.g. http://www.cs.toronto.edu/∼wkc/kmerHMM.

Screening and characterization of a Annenix A2 binding aptamer that inhibits the proliferation of myeloma cells.

PubMed

Zhou, Weihua; Zhang, Yibin; Zeng, Yayue; Peng, Minyuan; Li, Hui; Sun, Shuming; Ma, Bianying; Wang, Yanpeng; Ye, Mao; Liu, Jing

2018-06-12

Multiple myeloma (MM) is a malignant plasma cell disease and is considered incurable. Annexin A2 (ANXA2) is closely related to the proliferation and adhesion of MM. Using protein-SELEX, we performed a screen for aptamers that bind GST-ANXA2 from a library, and GST protein was used for negative selection. The enrichment of the ssDNA pool was monitored by filter-binding assay during selection. After nine rounds of screening and high-throughput sequencing, we obtained six candidate aptamers that bind to the ANXA2 protein. The affinities of the candidate aptamers for ANXA2 were determined by ELONA. Binding of aptamer wh6 to the ANXA2 protein and to the MM cell was verified by aptamer pulldown experiment and flow cytometry, respectively. Aptamer wh6 binds the ANXA2 protein with good stability and has a dissociation constant in the nanomolar range. The binding specificity of aptamer wh6 was confirmed in vivo in nude mouse xenografts with MM cells and with MM bone marrow aspirates. Furthermore, aptamer wh6 can block MM cell adhesion to ANXA2 and block the proliferation of MM cells induced by ANXA2. In summary, wh6 can be considered a promising candidate tool for MM diagnosis and treatment. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication.

PubMed

Abdul Ahmad, Siti Aisyah; Palanisamy, Uma D; Tejo, Bimo A; Chew, Miaw Fang; Tham, Hong Wai; Syed Hassan, Sharifah

2017-11-21

The rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated. Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC 50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay. Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC 50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity. Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

Accurate ensemble molecular dynamics binding free energy ranking of multidrug-resistant HIV-1 proteases.

PubMed

Sadiq, S Kashif; Wright, David W; Kenway, Owain A; Coveney, Peter V

2010-05-24

Accurate calculation of important thermodynamic properties, such as macromolecular binding free energies, is one of the principal goals of molecular dynamics simulations. However, single long simulation frequently produces incorrectly converged quantitative results due to inadequate sampling of conformational space in a feasible wall-clock time. Multiple short (ensemble) simulations have been shown to explore conformational space more effectively than single long simulations, but the two methods have not yet been thermodynamically compared. Here we show that, for end-state binding free energy determination methods, ensemble simulations exhibit significantly enhanced thermodynamic sampling over single long simulations and result in accurate and converged relative binding free energies that are reproducible to within 0.5 kcal/mol. Completely correct ranking is obtained for six HIV-1 protease variants bound to lopinavir with a correlation coefficient of 0.89 and a mean relative deviation from experiment of 0.9 kcal/mol. Multidrug resistance to lopinavir is enthalpically driven and increases through a decrease in the protein-ligand van der Waals interaction, principally due to the V82A/I84V mutation, and an increase in net electrostatic repulsion due to water-mediated disruption of protein-ligand interactions in the catalytic region. Furthermore, we correctly rank, to within 1 kcal/mol of experiment, the substantially increased chemical potency of lopinavir binding to the wild-type protease compared to saquinavir and show that lopinavir takes advantage of a decreased net electrostatic repulsion to confer enhanced binding. Our approach is dependent on the combined use of petascale computing resources and on an automated simulation workflow to attain the required level of sampling and turn around time to obtain the results, which can be as little as three days. This level of performance promotes integration of such methodology with clinical decision support systems for the optimization of patient-specific therapy.

Identification of the bombesin receptor on murine and human cells by cross-linking experiments.

PubMed

Kris, R M; Hazan, R; Villines, J; Moody, T W; Schlessinger, J

1987-08-15

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

PubMed

Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

2013-09-17

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

The feasibility of an efficient drug design method with high-performance computers.

PubMed

Yamashita, Takefumi; Ueda, Akihiko; Mitsui, Takashi; Tomonaga, Atsushi; Matsumoto, Shunji; Kodama, Tatsuhiko; Fujitani, Hideaki

2015-01-01

In this study, we propose a supercomputer-assisted drug design approach involving all-atom molecular dynamics (MD)-based binding free energy prediction after the traditional design/selection step. Because this prediction is more accurate than the empirical binding affinity scoring of the traditional approach, the compounds selected by the MD-based prediction should be better drug candidates. In this study, we discuss the applicability of the new approach using two examples. Although the MD-based binding free energy prediction has a huge computational cost, it is feasible with the latest 10 petaflop-scale computer. The supercomputer-assisted drug design approach also involves two important feedback procedures: The first feedback is generated from the MD-based binding free energy prediction step to the drug design step. While the experimental feedback usually provides binding affinities of tens of compounds at one time, the supercomputer allows us to simultaneously obtain the binding free energies of hundreds of compounds. Because the number of calculated binding free energies is sufficiently large, the compounds can be classified into different categories whose properties will aid in the design of the next generation of drug candidates. The second feedback, which occurs from the experiments to the MD simulations, is important to validate the simulation parameters. To demonstrate this, we compare the binding free energies calculated with various force fields to the experimental ones. The results indicate that the prediction will not be very successful, if we use an inaccurate force field. By improving/validating such simulation parameters, the next prediction can be made more accurate.

Factors governing the substitution of La3+ for Ca2+ and Mg2+ in metalloproteins: a DFT/CDM study.

PubMed

Dudev, Todor; Chang, Li-Ying; Lim, Carmay

2005-03-23

Trivalent lanthanide cations are extensively being used in biochemical experiments to probe various dication-binding sites in proteins; however, the factors governing the binding specificity of lanthanide cations for these binding sites remain unclear. Hence, we have performed systematic studies to evaluate the interactions between La3+ and model Ca2+ - and Mg2+ -binding sites using density functional theory combined with continuum dielectric methods. The calculations reveal the key factors and corresponding physical bases favoring the substitution of trivalent lanthanides for divalent Ca2+ and Mg2+ in holoproteins. Replacing Ca2+ or Mg2+ with La3+ is facilitated by (1) minimizing the solvent exposure and the flexibility of the metal-binding cavity, (2) freeing both carboxylate oxygen atoms of Asp/Glu side chains in the metal-binding site so that they could bind bidentately to La3+, (3) maximizing the number of metal-bound carboxylate groups in buried sites, but minimizing the number of metal-bound carboxylate groups in solvent-exposed sites, and (4) including an Asn/Gln side chain for sites lined with four Asp/Glu side chains. In proteins bound to both Mg2+ and Ca2+, La3+ would prefer to replace Ca2+, as compared to Mg2+. A second Mg2+-binding site with a net positive charge would hamper the Mg2+ --> La3+ exchange, as compared to the respective mononuclear site, although the La3+ substitution of the first native metal is more favorable than the second one. The findings of this work are in accord with available experimental data.

Computational investigation of the binding mode of bis(hydroxylphenyl)arenes in 17β-HSD1: molecular dynamics simulations, MM-PBSA free energy calculations, and molecular electrostatic potential maps

NASA Astrophysics Data System (ADS)

Negri, Matthias; Recanatini, Maurizio; Hartmann, Rolf W.

2011-09-01

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the last step of the estrogen biosynthesis, namely the reduction of estrone to the biologically potent estradiol. As such it is a potentially attractive drug target for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. 17β-HSD1 belongs to the bisubstrate enzymes and exists as an ensemble of conformations. These principally differ in the region of the βFαG'-loop, suggesting a prominent role in substrate and inhibitor binding. Although several classes of potent non-steroidal 17β-HSD1 inhibitors currently exist, their binding mode is still unclear. We aimed to elucidate the binding mode of bis(hydroxyphenyl)arenes, a highly potent class of 17β-HSD1 inhibitors, and to rank these compounds correctly with respect to their inhibitory potency, two essential aspects in drug design. Ensemble docking experiments resulted in a steroidal binding mode for the closed enzyme conformations and in an alternative mode for the opened and occluded conformers with the inhibitors placed below the NADPH interacting with it synergically via π-π stacking and H-bond formation. Both binding modes were investigated by MD simulations and MM-PBSA binding free energy estimations using as representative member for this class compound 1 (50 nM). Notably, only the alternative binding mode proved stable and was energetically more favorable, while when simulated in the steroidal binding mode compound 1 was displaced from the active site. In parallel, ab initio studies of small NADPH-inhibitor complexes were performed, which supported the importance of the synergistic interaction between inhibitors and cofactor.

Identification of the DNA-Binding Domains of Human Replication Protein A That Recognize G-Quadruplex DNA

PubMed Central

Prakash, Aishwarya; Natarajan, Amarnath; Marky, Luis A.; Ouellette, Michel M.; Borgstahl, Gloria E. O.

2011-01-01

Replication protein A (RPA), a key player in DNA metabolism, has 6 single-stranded DNA-(ssDNA-) binding domains (DBDs) A-F. SELEX experiments with the DBDs-C, -D, and -E retrieve a 20-nt G-quadruplex forming sequence. Binding studies show that RPA-DE binds preferentially to the G-quadruplex DNA, a unique preference not observed with other RPA constructs. Circular dichroism experiments show that RPA-CDE-core can unfold the G-quadruplex while RPA-DE stabilizes it. Binding studies show that RPA-C binds pyrimidine- and purine-rich sequences similarly. This difference between RPA-C and RPA-DE binding was also indicated by the inability of RPA-CDE-core to unfold an oligonucleotide containing a TC-region 5′ to the G-quadruplex. Molecular modeling studies of RPA-DE and telomere-binding proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA-DE and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties. PMID:21772997

Feature integration and spatial attention: common processes for endogenous and exogenous orienting.

PubMed

Henderickx, David; Maetens, Kathleen; Soetens, Eric

2010-05-01

Briand (J Exp Psychol Hum Percept Perform 24:1243-1256, 1998) and Briand and Klein (J Exp Psychol Hum Percept Perform 13:228-241, 1987) demonstrated that spatial cueing effects are larger for detecting conjunction of features than for detecting simple features when spatial attention is oriented exogenously, and not when attention is oriented endogenously. Their results were interpreted as if only exogenous attention affects the posterior spatial attention system that performs the feature binding function attributed to spatial attention by Treisman's feature integration theory (FIT; 1980). In a series of 6 experiments, we attempted to replicate Briand's findings. Manipulations of distractor string size and symmetry of stimulus presentation left and right from fixation were implemented in Posner's cueing paradigm. The data indicate that both exogenous and endogenous cueing address the same attentional mechanism needed for feature binding. The results also limit the generalisability of Briand's proposal concerning the role of exogenous attention in feature integration. Furthermore, the importance to control the effect of unintended attentional capture in a cueing task is demonstrated.

Predicting protein-binding RNA nucleotides with consideration of binding partners.

PubMed

Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

2015-06-01

In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in most performance measures. To the best of our knowledge, this is the first sequence-based prediction of protein-binding nucleotides in RNA which considers the binding partner of RNA. The new model will provide valuable information for designing biochemical experiments to find putative protein-binding sites in RNA with unknown structure. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The Effects of Extending of Co-planarity in a Series of Structurally Relative Polypyridyl Palladium(II) Complexes on DNA-binding and Cytotoxicity Properties

PubMed Central

Shahraki, Somaye; Mansouri-Torshizi, Hassan; Sori Nezami, Ziba; Ghahghaei, Arezou; Yaghoubi, Fatemeh; Divsalar, Adeleh; Saboury, Ali-Akbar; H. Shirazi, Farshad

2014-01-01

In depth interaction studies between calf thymus deoxyribonucleic acid (CT-DNA) and a series of four structurally relative palladium(II) complexes [Pd(en)(HB)](NO3)2 (a-d), where en is ethylenediamine and heterocyclic base (HB) is 2,2'-bipyridine (bpy, a); 1,10-phenanthroline (phen, b); dipyridoquinoxaline (dpq, c) and dipyridophenazine (dppz, d) (Figure 1), were performed. These studies have been investigated by utilizing the electronic absorption spectroscopy, fluorescence spectra and ethidium bromide (EBr) displacement and gel filtration techniques. a-d complexes cooperatively bind and denature the DNA at low concentrations. Their concentration at midpoint of transition, L1/2, follows the order a >> b > c > d. Also the g, the number of binding sites per 1000 nucleotides, follows the order a >> b ~ c > d. EBr and Scatchard experiments for a-d complexes suggest efficient intercalative binding affinity to CT-DNA giving the order: d > c > b > a. Several binding and thermodynamic parameters are also described. The biological activity of these cationic and water soluble palladium complexes were tested against chronic myelogenous leukemia cell line, K562. b, c and d complexes show cytotoxic concentration (Cc50) values much lower than cisplatin. PMID:25587317

Towards the elucidation of molecular determinants of cooperativity in the liver bile acid binding protein.

PubMed

Pedò, Massimo; D'Onofrio, Mariapina; Ferranti, Pasquale; Molinari, Henriette; Assfalg, Michael

2009-11-15

Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using (15)N-labelled ligand, while the relative populations of differently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein-observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein containing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore identified a mutation able to disrupt energetic communication functional to efficient binding and conclude that the buried histidine establishes contacts that stabilize the ternary complex. 2009 Wiley-Liss, Inc.

Dye-induced aggregation of single stranded RNA: a mechanistic approach.

PubMed

Biver, Tarita; Ciatto, Carlo; Secco, Fernando; Venturini, Marcella

2006-08-15

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 degrees C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PD(I) and an internal complex PD(II) form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (C(P)/C(D)). For C(P)/C(D)<2.5, cooperative formation of stacks external to polymer strands prevails (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g=1), equilibrium constant for the nucleation step (K( *)=(1.4+/-0.6)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(7)M(-1)s(-1), k(d)=1.1 x 10(2)s(-1)). Stopped-flow experiments, performed at low ionic strength (I=0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that C(P)/C(D)<2.5.

Na+/substrate Coupling in the Multidrug Antiporter NorM Probed with a Spin-labeled Substrate

PubMed Central

Steed, P. Ryan; Stein, Richard A.; Mishra, Smriti; Goodman, Michael C.; Mchaourab, Hassane S.

2013-01-01

NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na+ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal to characterize mechanistically relevant binding interactions with NorM and to directly address the coupling of ion and drug binding. Fluorescence and EPR experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na+. Using double electron-electron resonance (DEER) spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na+ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na+ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na+ gradient across the native membrane, govern Na+/drug coupling during antiport. PMID:23902581

A simple and efficient method to enhance audiovisual binding tendencies

PubMed Central

Wozny, David R.; Shams, Ladan

2017-01-01

Individuals vary in their tendency to bind signals from multiple senses. For the same set of sights and sounds, one individual may frequently integrate multisensory signals and experience a unified percept, whereas another individual may rarely bind them and often experience two distinct sensations. Thus, while this binding/integration tendency is specific to each individual, it is not clear how plastic this tendency is in adulthood, and how sensory experiences may cause it to change. Here, we conducted an exploratory investigation which provides evidence that (1) the brain’s tendency to bind in spatial perception is plastic, (2) that it can change following brief exposure to simple audiovisual stimuli, and (3) that exposure to temporally synchronous, spatially discrepant stimuli provides the most effective method to modify it. These results can inform current theories about how the brain updates its internal model of the surrounding sensory world, as well as future investigations seeking to increase integration tendencies. PMID:28462016

An Undergraduate Laboratory Experiment that Utilizes a Glass Fiber Filter Assay to Determine the Steroid Specificity and Equilibrium Binding Properties of Glucocorticoid Receptors.

ERIC Educational Resources Information Center

John, Nancy J.; Firestone, Gary L.

1987-01-01

Describes two complementary laboratory exercises that use the glass fiber assay to assess receptor specificity and hormone binding affinity in rat liver cytoplasmic extracts. Details the methods, materials and protocol of the experiments. Discusses the basic concepts illustrated and the feasibility of using the experiments at the undergraduate…

Micro-chromatin Immunoprecipation (μChIP) Protocol for Real-time PCR Analysis of a Limited Amount of Cells.

PubMed

Gillotin, Sébastien; Guillemot, François

2016-06-20

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is an important strategy to study gene regulation. When availability of cells is limited, however, it can be useful to focus on specific genes to investigate in depth the role of transcription factors or histone marks. Unfortunately, performing ChIP experiments to study transcription factors' binding to DNA can be difficult when biological material is restricted. This protocol describes a robust method to perform μChIP for over-expressed or endogenous transcription factors using ~100,000 cells per ChIP experiment (Masserdotti et al ., 2015). We also describe optimization steps, which we think are critical for this protocol to work and which can be used to further reduce the number of cells.

Investigation on the interaction of Rutin with serum albumins: Insights from spectroscopic and molecular docking techniques.

PubMed

Sengupta, Priti; Sardar, Pinki Saha; Roy, Pritam; Dasgupta, Swagata; Bose, Adity

2018-06-01

The binding interaction of Rutin, a flavonoid, with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), were investigated using different spectroscopic techniques, such as fluorescence, time-resolved single photon counting (TCSPC) and circular dichroism (CD) spectroscopy as well as molecular docking method. The emission studies revealed that the fluorescence quenching of BSA/HSA by Rutin occurred through a simultaneous static and dynamic quenching process, and we have evaluated both the quenching constants individually. The binding constants of Rutin-BSA and Rutin-HSA system were found to be 2.14 × 10 6  M -1 and 2.36 × 10 6  M -1 at 298 K respectively, which were quite high. Further, influence of some biologically significant metal ions (Ca 2+ , Zn 2+ and Mg 2+ ) on binding of Rutin to BSA and HSA were also investigated. Thermodynamic parameters justified the involvement of hydrogen bonding and weak van der Waals forces in the interaction of Rutin with both BSA and HSA. Further a site-marker competitive experiment was performed to evaluate Rutin binding site in the albumins. Additionally, the CD spectra of BSA and HSA revealed that the secondary structure of the proteins was perturbed in the presence of Rutin. Finally protein-ligand docking studies have also been performed to determine the probable location of the ligand molecule. Copyright © 2018 Elsevier B.V. All rights reserved.

The effects of value on context-item associative memory in younger and older adults.

PubMed

Hennessee, Joseph P; Knowlton, Barbara J; Castel, Alan D

2018-02-01

Valuable items are often remembered better than items that are less valuable by both older and younger adults, but older adults typically show deficits in binding. Here, we examine whether value affects the quality of recognition memory and the binding of incidental details to valuable items. In Experiment 1, participants learned English words each associated with a point-value they earned for correct recognition with the goal of maximizing their score. In Experiment 2, value was manipulated by presenting items that were either congruent or incongruent with an imagined state of physiological need (e.g., hunger). In Experiment 1, point-value was associated with enhanced recollection in both age groups. Memory for the color associated with the word was in fact reduced for high-value recollected items compared with low-value recollected items, suggesting value selectively enhances binding of task-relevant details. In Experiment 2, memory for learned images was enhanced by value in both age groups. However, value differentially enhanced binding of an imagined context to the item in younger and older adults, with a strong trend for increased binding in younger adults only. These findings suggest that value enhances episodic encoding in both older and younger adults but that binding of associated details may be reduced for valuable items compared to less valuable items, particularly in older adults. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Great interactions: How binding incorrect partners can teach us about protein recognition and function

PubMed Central

Vamparys, Lydie; Laurent, Benoist; Carbone, Alessandra

2016-01-01

ABSTRACT Protein–protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross‐docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross‐docking predictions using the area under the specificity‐sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding‐site predictions resulting from the cross‐docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408–1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:27287388

Performance of MDockPP in CAPRI rounds 28-29 and 31-35 including the prediction of water-mediated interactions.

PubMed

Xu, Xianjin; Qiu, Liming; Yan, Chengfei; Ma, Zhiwei; Grinter, Sam Z; Zou, Xiaoqin

2017-03-01

Protein-protein interactions are either through direct contacts between two binding partners or mediated by structural waters. Both direct contacts and water-mediated interactions are crucial to the formation of a protein-protein complex. During the recent CAPRI rounds, a novel parallel searching strategy for predicting water-mediated interactions is introduced into our protein-protein docking method, MDockPP. Briefly, a FFT-based docking algorithm is employed in generating putative binding modes, and an iteratively derived statistical potential-based scoring function, ITScorePP, in conjunction with biological information is used to assess and rank the binding modes. Up to 10 binding modes are selected as the initial protein-protein complex structures for MD simulations in explicit solvent. Water molecules near the interface are clustered based on the snapshots extracted from independent equilibrated trajectories. Then, protein-ligand docking is employed for a parallel search for water molecules near the protein-protein interface. The water molecules generated by ligand docking and the clustered water molecules generated by MD simulations are merged, referred to as the predicted structural water molecules. Here, we report the performance of this protocol for CAPRI rounds 28-29 and 31-35 containing 20 valid docking targets and 11 scoring targets. In the docking experiments, we predicted correct binding modes for nine targets, including one high-accuracy, two medium-accuracy, and six acceptable predictions. Regarding the two targets for the prediction of water-mediated interactions, we achieved models ranked as "excellent" in accordance with the CAPRI evaluation criteria; one of these two targets is considered as a difficult target for structural water prediction. Proteins 2017; 85:424-434. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Utilization of ICP/OES for the determination of trace metal binding to different humic fractions.

PubMed

de la Rosa, G; Peralta-Videa, J R; Gardea-Torresdey, J L

2003-02-28

In this study, the use of inductively coupled plasma/optical emission spectrometry (ICP/OES) to determine multi-metal binding to three biomasses, Sphagnum peat moss, humin and humic acids is reported. All the investigations were performed under part per billion (ppb) concentrations. Batch pH profile experiments were performed using multi-metal solutions of Cd(II), Cu(II), Pb(II), Ni(II), Cr(III) and Cr(VI). The results showed that at pH 2 and 3, the metal affinity of the three biomasses exposed to the multi-metal solution that included Cr(III) presented the following order: Cu(II), Pb(II)>Ni(II)>Cr(III)>Cd(II). On the other hand, when Cr(VI) was in the heavy metal mixture, Sphagnum peat moss and humin showed the following affinity: Cu(II), Pb(II)>Ni(II)>Cr(VI)>Cd(II); however, the affinity of the humic acids was: Cu(II)>Pb(II), Cr(VI)>Ni(II)>Cd(II). The results demonstrated that pH values of 4 and 5 were the most favorable for the heavy metal binding process. At pH 5, all the metals, except for Cr(VI), were bound between 90 and 100% to the three biomasses. However, the binding capacity of humic acids decreased at pH 6 in the presence of Cr(VI). The results showed that the ICP/OES permits the determination of heavy metal binding to organic matter at ppb concentration. These results will be very useful in understanding the role of humic substances in the fate and transport of heavy metals, and thus could provide information to develop new methodologies for the removal of low concentrations of toxic heavy metals from contaminated waters.

Performance of HADDOCK and a simple contact-based protein-ligand binding affinity predictor in the D3R Grand Challenge 2

NASA Astrophysics Data System (ADS)

Kurkcuoglu, Zeynep; Koukos, Panagiotis I.; Citro, Nevia; Trellet, Mikael E.; Rodrigues, J. P. G. L. M.; Moreira, Irina S.; Roel-Touris, Jorge; Melquiond, Adrien S. J.; Geng, Cunliang; Schaarschmidt, Jörg; Xue, Li C.; Vangone, Anna; Bonvin, A. M. J. J.

2018-01-01

We present the performance of HADDOCK, our information-driven docking software, in the second edition of the D3R Grand Challenge. In this blind experiment, participants were requested to predict the structures and binding affinities of complexes between the Farnesoid X nuclear receptor and 102 different ligands. The models obtained in Stage1 with HADDOCK and ligand-specific protocol show an average ligand RMSD of 5.1 Å from the crystal structure. Only 6/35 targets were within 2.5 Å RMSD from the reference, which prompted us to investigate the limiting factors and revise our protocol for Stage2. The choice of the receptor conformation appeared to have the strongest influence on the results. Our Stage2 models were of higher quality (13 out of 35 were within 2.5 Å), with an average RMSD of 4.1 Å. The docking protocol was applied to all 102 ligands to generate poses for binding affinity prediction. We developed a modified version of our contact-based binding affinity predictor PRODIGY, using the number of interatomic contacts classified by their type and the intermolecular electrostatic energy. This simple structure-based binding affinity predictor shows a Kendall's Tau correlation of 0.37 in ranking the ligands (7th best out of 77 methods, 5th/25 groups). Those results were obtained from the average prediction over the top10 poses, irrespective of their similarity/correctness, underscoring the robustness of our simple predictor. This results in an enrichment factor of 2.5 compared to a random predictor for ranking ligands within the top 25%, making it a promising approach to identify lead compounds in virtual screening.

Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges

PubMed Central

Naiser, Thomas; Ehler, Oliver; Kayser, Jona; Mai, Timo; Michel, Wolfgang; Ott, Albrecht

2008-01-01

Background The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations. Results We performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths (in situations without competitive hybridization) in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect (MM or bulge) type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA/DNA and RNA/DNA duplexes shows, that RNA/DNA purine-purine MMs are more discriminating than corresponding DNA/DNA MMs. In DNA/DNA MM discrimination the affected base pair (C·G vs. A·T) is the pertinent parameter. We attribute these differences to the different structures of the duplexes (A vs. B form). Conclusion We have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role. PMID:18477387

Thiophene-based rhodamine as selectivef luorescence probe for Fe(III) and Al(III) in living cells.

PubMed

Wang, Kun-Peng; Chen, Ju-Peng; Zhang, Si-Jie; Lei, Yang; Zhong, Hua; Chen, Shaojin; Zhou, Xin-Hong; Hu, Zhi-Qiang

2017-09-01

The thiophene-modified rhodamine 6G (GYJ) has been synthesized as a novel chemosensor. The sensor has sufficiently high selectivity and sensitivity for the detection of Fe 3+ and Al 3+ ions (M 3+ ) by fluorescence and ultraviolet spectroscopy with a strong ability for anti-interference performance. The binding ratio of M 3+ -GYJ complex was determined to be 2:1 according to the Job's plot. The binding constants for Fe 3+ and Al 3+ were calculated to be 3.91 × 10 8 and 5.26 × 10 8  M -2 , respectively. All these unique features made it particularly favorable for cellular imaging applications. The obvious fluorescence microscopy experiments demonstrated that the probes could contribute to the detection of Fe 3+ and Al 3+ in related cells and biological organs with satisfying resolution. Graphical abstract GYJ has high selectivity and sensitivity for the detection of Fe(III) and Al(III) with the binding ratio of 2:1.

Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells

PubMed Central

Capuani, Fabrizio; Conte, Alexia; Argenzio, Elisabetta; Marchetti, Luca; Priami, Corrado; Polo, Simona; Di Fiore, Pier Paolo; Sigismund, Sara; Ciliberto, Andrea

2015-01-01

Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration. Here we use a simple modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR. While the threshold is remarkably robust, a more sophisticated model predicted that it could be modulated as a function of EGFR levels on the cell surface. We confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes—at the supraphysiological levels of receptor and/or ligand associated with cancer—uncoupling of the mechanisms leading to signalling through phosphorylation and attenuation through ubiquitination. PMID:26264748

PTB and TIAR binding to insulin mRNA 3'- and 5'UTRs; implications for insulin biosynthesis and messenger stability.

PubMed

Fred, Rikard G; Mehrabi, Syrina; Adams, Christopher M; Welsh, Nils

2016-09-01

Insulin expression is highly controlled on the posttranscriptional level. The RNA binding proteins (RBPs) responsible for this result are still largely unknown. To identify RBPs that bind to insulin mRNA we performed mass spectrometry analysis on proteins that bound synthetic oligonucloetides mimicing the 5'- and the 3'-untranslated regions (UTRs) of rat and human insulin mRNA in vitro . We observed that the RBPs heterogeneous nuclear ribonucleoprotein (hnRNP) U, polypyrimidine tract binding protein (PTB), hnRNP L and T-cell restricted intracellular antigen 1-related protein (TIA-1-related protein; TIAR) bind to insulin mRNA sequences, and that the in vitro binding affinity of these RBPs changed when INS-1 cells were exposed to glucose, 3-isobutyl-1-methylxanthine (IBMX) or nitric oxide. High glucose exposure resulted in a modest increase in PTB and TIAR binding to an insulin mRNA sequence. The inducer of nitrosative stress DETAnonoate increased markedly hnRNP U and TIAR mRNA binding. An increased PTB to TIAR binding ratio in vitro correlated with higher insulin mRNA levels and insulin biosynthesis rates in INS-1 cells. To further investigate the importance of RNA-binding proteins for insulin mRNA stability, we decreased INS-1 and EndoC-βH1 cell levels of PTB and TIAR by RNAi. In both cell lines, decreased levels of PTB resulted in lowered insulin mRNA levels while decreased levels of TIAR resulted in increased insulin mRNA levels. Thapsigargin-induced stress granule formation was associated with a redistribution of TIAR from the cytosol to stress granules. These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.

Experiments of the Essential Amino Acids at high temperature and high pressure using DAC

NASA Astrophysics Data System (ADS)

Kubo, K.; Okamoto, K.

2017-12-01

Amino acids are organic compounds that form the fundamental part of life. Proteins are formed by peptide binding and polymerization of amino acids. Amino acids are polymerized in the ridge hydrothermal field, formed proteins, and might be evolved into life. Experimental studies on the polymerization of amino acids in hydrothermal environments have been conducted. However, they were hydrothermal experiments and after the experiments. All run products (amid-acids) were observed at ambient condition. Few in-situ observations of amino acids were done in experiments in hydrothermal condition. In order to perform in-situ observation of the polymerization of amino acids, we have conducted the DAC experiments. Amino acids were filled in the DAC, pressures were applied, then heated to high temperature with Raman analysis. In preliminary experiment using glycine, polymerization forming diglycine, were completed. Investigation amino acids polymerization under hydrothermal condition would shed light for new view of early life science.

Graphene-Based Liquid-Gated Field Effect Transistor for Biosensing: Theory and Experiments

PubMed Central

Reiner-Rozman, Ciril; Larisika, Melanie; Nowak, Christoph; Knoll, Wolfgang

2015-01-01

We present an experimental and theoretical characterization for reduced Graphene-Oxide (rGO) based FETs used for biosensing applications. The presented approach shows a complete result analysis and theoretically predictable electrical properties. The formulation was tested for the analysis of the device performance in the liquid gate mode of operation with variation of the ionic strength and pH-values of the electrolytes in contact with the FET. The dependence on the Debye length was confirmed experimentally and theoretically, utilizing the Debye length as a working parameter and thus defining the limits of applicability for the presented rGO-FETs. Furthermore, the FETs were tested for the sensing of biomolecules (bovine serum albumin (BSA) as reference) binding to gate-immobilized anti-BSA antibodies and analyzed using the Langmuir binding theory for the description of the equilibrium surface coverage as a function of the bulk (analyte) concentration. The obtained binding coefficients for BSA are found to be same as in results from literature, hence confirming the applicability of the devices. The FETs used in the experiments were fabricated using wet-chemically synthesized graphene, displaying high electron and hole mobility (μ) and provide the strong sensitivity also for low potential changes (by change of pH, ion concentration, or molecule adsorption). The binding coefficient for BSA-anti-BSA interaction shows a behavior corresponding to the Langmuir adsorption theory with a Limit of Detection (LOD) in the picomolar concentration range. The presented approach shows high reproducibility and sensitivity and a good agreement of the experimental results with the calculated data. PMID:25791463

The Discovery of [Ni(NHC)RCN]2 Species and their Role as Cycloaddition Catalysts for the Formation of Pyridines

PubMed Central

Stolley, Ryan M.; Duong, Hung A.; Thomas, David R.; Louie, Janis

2012-01-01

The reaction of Ni(COD)2, IPr, and nitrile affords dimeric [Ni(IPr)RCN]2 in high yields. X-ray analysis revealed these species display simultaneous η1- and η2-nitrile binding modes. These dimers are catalytically competent in the formation of pyridines from the cycloaddition of diynes and nitriles. Kinetic analysis showed the reaction to be first order in [Ni(IPr)RCN]2, zeroth order in added IPr, zeroth order in nitrile, and zeroth order in diyne. Extensive stoichiometric competition studies were performed, and selective incorporation of the exogenous, not dimer bound, nitrile was observed. Post cycloaddition, the dimeric state was found to be largely preserved. Nitrile and ligand exchange experiments were performed and found to be inoperative in the catalytic cycle. These observations suggest a mechanism whereby the catalyst is activated by partial dimer-opening followed by binding of exogenous nitrile and subsequent oxidative heterocoupling. PMID:22917161

High performance wash-free magnetic bioassays through microfluidically enhanced particle specificity.

PubMed

Bechstein, Daniel J B; Lee, Jung-Rok; Ooi, Chin Chun; Gani, Adi W; Kim, Kyunglok; Wilson, Robert J; Wang, Shan X

2015-06-30

Magnetic biosensors have emerged as a sensitive and versatile platform for high performance medical diagnostics. These magnetic biosensors require well-tailored magnetic particles as detection probes, which need to give rise to a large and specific biological signal while showing very low nonspecific binding. This is especially important in wash-free bioassay protocols, which do not require removal of particles before measurement, often a necessity in point of care diagnostics. Here we show that magnetic interactions between magnetic particles and magnetized sensors dramatically impact particle transport and magnetic adhesion to the sensor surfaces. We investigate the dynamics of magnetic particles' biomolecular binding and magnetic adhesion to the sensor surface using microfluidic experiments. We elucidate how flow forces can inhibit magnetic adhesion, greatly diminishing or even eliminating nonspecific signals in wash-free magnetic bioassays, and enhancing signal to noise ratios by several orders of magnitude. Our method is useful for selecting and optimizing magnetic particles for a wide range of magnetic sensor platforms.

High performance wash-free magnetic bioassays through microfluidically enhanced particle specificity

PubMed Central

Bechstein, Daniel J.B.; Lee, Jung-Rok; Ooi, Chin Chun; Gani, Adi W.; Kim, Kyunglok; Wilson, Robert J.; Wang, Shan X.

2015-01-01

Magnetic biosensors have emerged as a sensitive and versatile platform for high performance medical diagnostics. These magnetic biosensors require well-tailored magnetic particles as detection probes, which need to give rise to a large and specific biological signal while showing very low nonspecific binding. This is especially important in wash-free bioassay protocols, which do not require removal of particles before measurement, often a necessity in point of care diagnostics. Here we show that magnetic interactions between magnetic particles and magnetized sensors dramatically impact particle transport and magnetic adhesion to the sensor surfaces. We investigate the dynamics of magnetic particles’ biomolecular binding and magnetic adhesion to the sensor surface using microfluidic experiments. We elucidate how flow forces can inhibit magnetic adhesion, greatly diminishing or even eliminating nonspecific signals in wash-free magnetic bioassays, and enhancing signal to noise ratios by several orders of magnitude. Our method is useful for selecting and optimizing magnetic particles for a wide range of magnetic sensor platforms. PMID:26123868

Scanpath memory binding: multiple read-out experiments

NASA Astrophysics Data System (ADS)

Stark, Lawrence W.; Privitera, Claudio M.; Yang, Huiyang; Azzariti, Michela; Ho, Yeuk F.; Chan, Angie; Krischer, Christof; Weinberger, Adam

1999-05-01

The scanpath theory proposed that an internal spatial- cognitive model controls perception and the active looking eye movements, EMs, of the scanpath sequence. Evidence for this came from new quantitative methods, experiments with ambiguous figures and visual imagery and from MRI studies, all on cooperating human subjects. Besides recording EMs, we introduce other experimental techniques wherein the subject must depend upon memory bindings as in visual imagery, but may call upon other motor behaviors than EMs to read-out the remembered patterns. How is the internal model distributed and operationally assembled. The concept of binding speaks to the assigning of values for the model and its execution in various parts of the brain. Current neurological information helps to localize different aspects of the spatial-cognitive model in the brain. We suppose that there are several levels of 'binding' -- semantic or symbolic binding, structural binding for the spatial locations of the regions-of-interest and sequential binding for the dynamic execution program that yields the sequence of EMs. Our aim is to dissect out respective contributions of these different forms of binding.

The Solution Structure, Binding Properties, and Dynamics of the Bacterial Siderophore-binding Protein FepB*

PubMed Central

Chu, Byron C. H.; Otten, Renee; Krewulak, Karla D.; Mulder, Frans A. A.; Vogel, Hans J.

2014-01-01

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs. PMID:25173704

Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS.

PubMed

Martí-Arbona, Ricardo; Teshima, Munehiro; Anderson, Penelope S; Nowak-Lovato, Kristy L; Hong-Geller, Elizabeth; Unkefer, Clifford J; Unkefer, Pat J

2012-01-01

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding. Copyright © 2012 S. Karger AG, Basel.

Hemoadsorption removes tumor necrosis factor, interleukin-6, and interleukin-10, reduces nuclear factor-kappaB DNA binding, and improves short-term survival in lethal endotoxemia.

PubMed

Kellum, John A; Song, Mingchen; Venkataraman, Ramesh

2004-03-01

Previous studies have shown that inflammatory mediators can be removed from the circulation with hemofiltration and that adsorption plays an important role. Because adsorptive capacity of hollow-fiber dialyzers is limited, we sought to determine whether hemoadsorption using high surface area beads would result in greater mediator removal and improved survival in experimental sepsis. Randomized controlled laboratory experiment. University laboratory. Sixty-six adult Sprague-Dawley rats. We conducted two ex vivo and two in vivo experiments. For in vivo experiments, we administered Escherichia coli endotoxin (20 mg/kg) by intravenous infusion and then randomized each animal to receive either hemoadsorption or a sham circuit for 4 hrs. Hemoadsorption was performed for 4 hrs using an arterial-venous circuit and a CytoSorb cartridge containing 10 g of polystyrene divinyl benzene copolymer beads with a biocompatible polyvinylpyrrolidone coating. Survival time was measured to a maximum of 12 hrs. In a separate set of experiments, we studied 12 animals using the same protocol except that we killed all animals at 4 hrs and removed standardized sections of liver for analysis of nuclear factor-kappaB DNA binding. Mean survival time among hemoadsorption-treated animals was 629+/-114 vs. 518+/-120 mins for sham-treated animals (p <.01). Overall survival (defined at 12 hrs) was also significantly better in the hemoadsorption group, seven of 20 vs. one of 20 (p <.05). Plasma interleukin-6 and interleukin-10 concentrations and liver nuclear factor-kappaB DNA binding were significantly reduced by hemoadsorption. Ex vivo experiments showed no endotoxin adsorption but strengthened our in vivo observations by showing rapid adsorption of tumor necrosis factor, interleukin-6, and interleukin-10. Hemoadsorption was associated with reduced inflammation and improved survival in this murine model of septic shock.

Solution structure of the antitermination protein NusB of Escherichia coli: a novel all-helical fold for an RNA-binding protein.

PubMed Central

Huenges, M; Rölz, C; Gschwind, R; Peteranderl, R; Berglechner, F; Richter, G; Bacher, A; Kessler, H; Gemmecker, G

1998-01-01

The NusB protein of Escherichia coli is involved in the regulation of rRNA biosynthesis by transcriptional antitermination. In cooperation with several other proteins, it binds to a dodecamer motif designated rrn boxA on the nascent rRNA. The antitermination proteins of E.coli are recruited in the replication cycle of bacteriophage lambda, where they play an important role in switching from the lysogenic to the lytic cycle. Multidimensional heteronuclear NMR experiments were performed with recombinant NusB protein labelled with 13C, 15N and 2H. The three-dimensional structure of the protein was solved from 1926 NMR-derived distances and 80 torsion angle restraints. The protein folds into an alpha/alpha-helical topology consisting of six helices; the arginine-rich N-terminus appears to be disordered. Complexation of the protein with an RNA dodecamer equivalent to the rrn boxA site results in chemical shift changes of numerous amide signals. The overall packing of the protein appears to be conserved, but the flexible N-terminus adopts a more rigid structure upon RNA binding, indicating that the N-terminus functions as an arginine-rich RNA-binding motif (ARM). PMID:9670024

Ultralong-range Rydberg Molecules: Investigation of a Novel Binding Mechanism

NASA Astrophysics Data System (ADS)

Butscher, Björn; Bendkowsky, Vera; Nipper, Johannes; Balewski, Jonathan; Shaffer, James P.; Löw, Robert; Pfau, Tilman

2010-03-01

For highly excited Rydberg atoms, the scattering of the Rydberg electron from a nearby polarizable ground state atom can generate an attractive mean-field potential which is able to bind the ground state atom to the Rydberg atom within the Rydberg electron wave function at binding energies ranging from a few MHz to hundreds of MHz[1]. We present spectroscopic data on the observation of various bound states including the vibrational ground and excited states of rubidium dimers Rb(5S)-Rb(nS) as well as those of trimer states. Furthermore, we show calculations that reproduce the observed binding energies remarkably well and reveal that some of the excited states are purely bound by quantum reflection at a shape resonance for p-wave scattering [2]. To further characterize the coherent excitation of the molecular states, we performed echo experiments. [0pt] [1] V. Bendkowsky, B. Butscher, J. Nipper, J. P. Shaffer, R. Löw, T. Pfau, Nature 458, 1005 (2009); [2] V. Bendkowsky, B. Butscher, J. Nipper, J. Balewski, J. P. Shaffer, R. Löw, T. Pfau, W. Li, J. Stanojevic, T. Pohl,and J. M. Rost, arXiv:0912.4058 (2009)

Dynamics and lithium binding energies of polyelectrolytes based on functionalized poly(para-phenylene terephthalamide).

PubMed

Grozema, F C; Best, A S; van Eijck, L; Stride, J; Kearley, G J; de Leeuw, S W; Picken, S J

2005-04-28

Polyelectrolyte materials are an interesting class of electrolytes for use in fuel cell and battery applications. Poly(para-phenylene terephthalamide) (PPTA, Kevlar) is a liquid crystalline polymer that, when sulfonated, is a polyelectrolyte that exhibits moderate ion conductivity at elevated temperatures. In this work, quasi-elastic neutron scattering (QENS) experiments were performed to gain insight into the effect of the presence of lithium counterions on the chain dynamics in the material. It was found that the addition of lithium ions decreases the dynamics of the chains. Additionally, the binding of lithium ions to the sulfonic acids groups was investigated by density functional theory (DFT) calculations. It was found that the local surroundings of the sulfonic acid group have very little effect on the lithium-ion binding energy. Binding energies for a variety of different systems were all calculated to be around 150 kcal/mol. The DFT calculations also show the existence of a structure in which a single lithium ion interacts with two sulfonic acid moieties on different chains. The formation of such "electrostatic cross-links" is believed to be the source of the increased tendency to aggregate and the reduced dynamics in the presence of lithium ions.

Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases as antimitotic agents.

PubMed

Shaik, Thokhir B; Hussaini, S M Ali; Nayak, V Lakshma; Sucharitha, M Lakshmi; Malik, M Shaheer; Kamal, Ahmed

2017-06-01

Based on our previous results and literature precedence, a series of 2-anilinopyridinyl-benzothiazole Schiff bases were rationally designed by performing molecular modeling experiments on some selected molecules. The binding energies of the docked molecules were better than the E7010, and the Schiff base with trimethoxy group on benzothiazole moiety, 4y was the best. This was followed by the synthesis of a series of the designed molecules by a convenient synthetic route and evaluation of their anticancer potential. Most of the compounds have shown significant growth inhibition against the tested cell lines and the compound 4y exhibited good antiproliferative activity with a GI 50 value of 3.8µM specifically against the cell line DU145. In agreement with the docking results, 4y exerted cytotoxicity by the disruption of the microtubule dynamics by inhibiting tubulin polymerization via effective binding into colchicine domain, comparable to E7010. Detailed binding modes of 4y with colchicine binding site of tubulin were studied by molecular docking. Furthermore, 4y induced apoptosis as evidenced by biological studies like mitochondrial membrane potential, caspase-3, and Annexin V-FITC assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

Predicting DNA binding proteins using support vector machine with hybrid fractal features.

PubMed

Niu, Xiao-Hui; Hu, Xue-Hai; Shi, Feng; Xia, Jing-Bo

2014-02-21

DNA-binding proteins play a vitally important role in many biological processes. Prediction of DNA-binding proteins from amino acid sequence is a significant but not fairly resolved scientific problem. Chaos game representation (CGR) investigates the patterns hidden in protein sequences, and visually reveals previously unknown structure. Fractal dimensions (FD) are good tools to measure sizes of complex, highly irregular geometric objects. In order to extract the intrinsic correlation with DNA-binding property from protein sequences, CGR algorithm, fractal dimension and amino acid composition are applied to formulate the numerical features of protein samples in this paper. Seven groups of features are extracted, which can be computed directly from the primary sequence, and each group is evaluated by the 10-fold cross-validation test and Jackknife test. Comparing the results of numerical experiments, the group of amino acid composition and fractal dimension (21-dimension vector) gets the best result, the average accuracy is 81.82% and average Matthew's correlation coefficient (MCC) is 0.6017. This resulting predictor is also compared with existing method DNA-Prot and shows better performances. © 2013 The Authors. Published by Elsevier Ltd All rights reserved.

Molecular Features of the Copper Binding Sites in the Octarepeat Domain of the Prion Protein†

PubMed Central

Burns, Colin S.; Aronoff-Spencer, Eliah; Dunham, Christine M.; Lario, Paula; Avdievich, Nikolai I.; Antholine, William E.; Olmstead, Marilyn M.; Vrielink, Alice; Gerfen, Gary J.; Peisach, Jack; Scott, William G.; Millhauser, Glenn L.

2010-01-01

Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60–91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760–13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23–28, 57–91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly–Cu linkage is unstable below pH ≈6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form. PMID:11900542

Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

PubMed

Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

2007-11-01

We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

Effects of milk proteins on sperm binding to the zona pellucida and intracellular Ca(2+) concentration in stallion sperm.

PubMed

Coutinho da Silva, Marco A; Seidel, George E; Squires, Edward L; Graham, James K; Carnevale, Elaine M

2014-11-10

Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 compared with 2mM Ca(2+) resulted in greater (P<0.05) influx of Ca(2+) into sperm. In Experiment II, sperm incubated in media containing 1mg/mL of native phosphocaseinate (NP) or sodium caseinate (SC) showed similar baseline intracellular Ca(2+) and influx of Ca(2+) than control (TALP). In Experiment III, sperm-ZP binding assays were performed in TALP medium containing: no additions (TALP); 1mg/mL SC; 1 or 3mg/mL of α-casein; 1 or 3mg/mL of β-casein; and 1 or 3mg/mL of κ-casein. The number of stallion sperm bound to bovine ZP was greatest (P<0.05) when SC was used. Co-incubation in media containing single caseins (α-, β- or κ-casein) resulted in similar results to TALP; however, a dose effect (P<0.05) was observed for β- and κ-caseins. In conclusion, extracellular Ca(2+) concentration and milk proteins did not affect baseline intracellular calcium in stallion sperm. It appears that β- and κ-caseins may be responsible for enhancing sperm binding to ZP, but the mechanism remains unknown. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential binding of colors to objects in memory: red and yellow stick better than blue and green

PubMed Central

Kuhbandner, Christof; Spitzer, Bernhard; Lichtenfeld, Stephanie; Pekrun, Reinhard

2015-01-01

Both evolutionary considerations and recent research suggest that the color red serves as a signal indicating an object’s importance. However, until now, there is no evidence that this signaling function of red is also reflected in human memory. To examine the effect of red on memory, we conducted four experiments in which we presented objects colored in four different colors (red, green, blue, and yellow) and measured later memory for the presence of an object and for the color of an object. Across experiments, we varied the type of objects (words vs. pictures), task complexity (single objects vs. multiple objects in visual scenes), and intentionality of encoding (intentional vs. incidental learning). Memory for the presence of an object was not influenced by color. However, in all four experiments, memory for the color of an object depended on color type and was particularly high for red and yellow-colored objects and particularly low for green-colored objects, indicating that the binding of colors into object memory representations varies as a function of color type. Analyzing the observers’ confidence in their color memories revealed that color not only influenced objective memory performance but also subjective confidence. Subjective confidence judgments differentiated well between correct and incorrect color memories for red-colored objects, but poorly for green-colored objects. Our findings reveal a previously unknown color effect which may be of considerable interest for both basic color research and applied settings like eyewitness testimony in which memory for color features is relevant. Furthermore, our results indicate that feature binding in memory is not a uniform process by which any attended feature is automatically bound into unitary memory representations. Rather, memory binding seems to vary across different subtypes of features, a finding that supports recent research showing that object features are stored in memory rather independently from each other. PMID:25784892

Differential binding of colors to objects in memory: red and yellow stick better than blue and green.

PubMed

Kuhbandner, Christof; Spitzer, Bernhard; Lichtenfeld, Stephanie; Pekrun, Reinhard

2015-01-01

Both evolutionary considerations and recent research suggest that the color red serves as a signal indicating an object's importance. However, until now, there is no evidence that this signaling function of red is also reflected in human memory. To examine the effect of red on memory, we conducted four experiments in which we presented objects colored in four different colors (red, green, blue, and yellow) and measured later memory for the presence of an object and for the color of an object. Across experiments, we varied the type of objects (words vs. pictures), task complexity (single objects vs. multiple objects in visual scenes), and intentionality of encoding (intentional vs. incidental learning). Memory for the presence of an object was not influenced by color. However, in all four experiments, memory for the color of an object depended on color type and was particularly high for red and yellow-colored objects and particularly low for green-colored objects, indicating that the binding of colors into object memory representations varies as a function of color type. Analyzing the observers' confidence in their color memories revealed that color not only influenced objective memory performance but also subjective confidence. Subjective confidence judgments differentiated well between correct and incorrect color memories for red-colored objects, but poorly for green-colored objects. Our findings reveal a previously unknown color effect which may be of considerable interest for both basic color research and applied settings like eyewitness testimony in which memory for color features is relevant. Furthermore, our results indicate that feature binding in memory is not a uniform process by which any attended feature is automatically bound into unitary memory representations. Rather, memory binding seems to vary across different subtypes of features, a finding that supports recent research showing that object features are stored in memory rather independently from each other.

Determination of thermodynamic parameters for complexation of calcium and magnesium with chondroitin sulfate isomers using isothermal titration calorimetry: Implications for calcium kidney-stone research

NASA Astrophysics Data System (ADS)

Rodgers, Allen L.; Jackson, Graham E.

2017-04-01

Chondroitin sulfate (CS) occurs in human urine. It has several potential binding sites for calcium and as such may play an inhibitory role in calcium oxalate and calcium phosphate (kidney stone disease by reducing the supersaturation (SS) and crystallization of these salts. Urinary magnesium is also a role player in determining speciation in stone forming processes. This study was undertaken to determine the thermodynamic parameters for binding of the disaccharide unit of two different CS isomers with calcium and magnesium. These included the binding constant K. Experiments were performed using an isothermal titration calorimeter (ITC) at 3 different pH levels in the physiological range in human urine. Data showed that interactions between the CS isomers and calcium and magnesium occur via one binding site, thought to be sulfate, and that log K values are 1.17-1.93 and 1.77-1.80 for these two metals respectively. Binding was significantly stronger in Mg-CS than in Ca-CS complexes and was found to be dependent on pH in the latter but not in the former. Furthermore, binding in Ca-CS complexes was dependent on the location of the sulfate binding site. This was not the case in the Mg-CS complexes. Interactions were shown to be entropy driven and enthalpy unfavourable. These findings can be used in computational modeling studies to predict the effects of the calcium and magnesium CS complexes on the speciation of calcium and the SS of calcium salts in real urine samples.

A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein

NASA Technical Reports Server (NTRS)

Salins, L. L.; Ware, R. A.; Ensor, C. M.; Daunert, S.

2001-01-01

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible. Copyright 2001 Academic Press.

Protein docking prediction using predicted protein-protein interface.

PubMed

Li, Bin; Kihara, Daisuke

2012-01-10

Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm), is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding

PubMed Central

Gilad, Yoav; Pritchard, Jonathan K.; Stephens, Matthew

2015-01-01

Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede. PMID:26406244

msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

PubMed

Raj, Anil; Shim, Heejung; Gilad, Yoav; Pritchard, Jonathan K; Stephens, Matthew

2015-01-01

Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

PubMed Central

Fjellström, Ola; Akkaya, Sibel; Beisel, Hans-Georg; Eriksson, Per-Olof; Erixon, Karl; Gustafsson, David; Jurva, Ulrik; Kang, Daiwu; Karis, David; Knecht, Wolfgang; Nerme, Viveca; Nilsson, Ingemar; Olsson, Thomas; Redzic, Alma; Roth, Robert; Sandmark, Jenny; Tigerström, Anna; Öster, Linda

2015-01-01

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds. PMID:25629509

The Determination of Vitamin D-Dependent Calcium Binding Protein in Chick Intesting: An Undergraduate Biochemistry Laboratory Experiment.

ERIC Educational Resources Information Center

Lessard, George M.

1980-01-01

Described is an experiment used in an undergraduate biochemistry laboratory involving inducing rickets in chicks and correlating the disease to a reduction in vitamin D-dependent calcium binding protein. Techniques involved are hormone induction, protein isolation, and radioisotope methodology. (Author/DS)

Shape coexistence and shape transition in light nuclei

NASA Astrophysics Data System (ADS)

Saxena, G.; Kumawat, M.; Singh, U. K.; Kaushik, M.; Jain, S. K.

2018-05-01

A systematic study has been performed to investigate the shape coexistence and shape transition for even-even nuclei between Z = 10-20 by employing Relativistic Mean-Filed plus BCS (RMF+BCS) approach. We calculate ground state properties viz. binding energy, deformation etc. for even-even nuclei to find the shape coexistence and shape transition. These results are found in agreement of recent experiments and consistent with other parameters of RMF and other theories.

A spectroscopic and molecular docking approach on the binding of tinzaparin sodium with human serum albumin

NASA Astrophysics Data System (ADS)

Abdullah, Saleh M. S.; Fatma, Sana; Rabbani, Gulam; Ashraf, Jalaluddin M.

2017-01-01

Protein bound toxins are poorly removed by conventional extracorporeal therapies. Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients with cancer. The interaction between tinzaparin, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by tinzaparin (TP). The binding constants and binding stoichiometry can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG° reveals that the binding process is a spontaneous process. Thermodynamic analysis shows that the HSA-TP complex formation occurs via hydrogen bonds, hydrophobic interactions and undergoes slight structural changes as evident by far-UV CD. It indicated that the hydrophobic interactions play a main role in the binding of TP to human serum albumin. In addition, the distance between TP (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 2.21 nm according to the Förster's resonance energy transfer theory. For the deeper understanding of the interaction, thermodynamic, and molecular docking studies were performed as well. Our docking results suggest that TP forms stable complex with HSA (Kb ∼ 104) and its primary binding site is located in subdomain IIA (Sudlow Site I). The results obtained herein will be of biological significance in pharmacology and clinical medicine.

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

PubMed Central

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

2012-01-01

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

Structure Determination of Ornithine-Linked Cisplatin by Infrared Multiple Photon Dissociation Action Spectroscopy

NASA Astrophysics Data System (ADS)

He, Chenchen; Kimutai, Bett; Hamlow, Lucas; Roy, Harrison; Nei, Y.-W.; Bao, Xun; Gao, Juehan; Martens, Jonathan K.; Berden, Giel; Oomens, Jos; Maitre, Philippe; Steinmetz, Vincent; McNary, Christopher P.; Armentrout, Peter B.; Chow, C. S.; Rodgers, M. T.

2016-06-01

Cisplatin [(NH_3)_2PtCl_2], the first FDA-approved platinum-based anticancer drug, has been widely used in cancer chemotherapy. Its pharmacological mechanism has been identified as its ability to coordinate to genomic DNA with guanine as its major target. Amino acid-linked cisplatin derivatives are being investigated as alternatives for cisplatin that may exhibit altered binding selectivity such as that found for ornithine-linked cisplatin (Ornplatin, [(Orn)PtCl_2]), which exhibits a preference for adenine over guanine in RNA. Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and complementary electronic structure calculations are performed on a series of Ornplatin complexes to elucidate the nature of binding of the Orn amino acid to the Pt center and how that binding is influenced by the local environment. The complexes examined in the work include: [(Orn-H)PtCl_2]-, [(Orn)PtCl]+, [(Orn)Pt(H_2O)Cl]+, and [(Orn)PtCl_2+Na]+. In contrast to that found previously for the glycine-linked cisplatin complex (Glyplatin), which binds via the backbone amino and carboxylate groups, binding of Orn in these complexes is found to involve both the backbone and sidechain amino groups. Extensive broadening of the IRMPD spectrum for the [(Orn)Pt(H_2O)Cl]+ complex suggests that either multiple structures are contributing to the measured spectrum or strong intra-molecular hydrogen-binding interactions are present. The results for Ornplatin lead to an interesting discussion about the differences in selectivity and reactivity versus cisplatin.

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes

NASA Astrophysics Data System (ADS)

Weinberger, Andreas; Walter, Vivien; MacEwan, Sarah R.; Schmatko, Tatiana; Muller, Pierre; Schroder, André P.; Chilkoti, Ashutosh; Marques, Carlos M.

2017-03-01

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

[Ceruloplasmin receptor on human erythrocytes].

PubMed

Saenko, E L; Basevich, V V; Iaropolov, A I

1988-08-01

The structural fragments of the human ceruloplasmin (CP) molecule and of erythrocyte receptors which provide for the specific interaction of CP with erythrocytes were identified, and their properties were investigated. The interaction of CP with erythrocytes, both intact and treated with neuroaminidase and proteolytic enzymes (trypsin, chymotrypsin, papaine, pronase E) is described. Experiments with CP reception were performed at 4 degrees C, using [125I]CP and [125I]asialo-CP. The parameters of binding were determined in Scatchard plots. It was demonstrated that the specific binding of CP to erythrocyte receptors is determined by its interaction with two structural sites of the carbohydrate moiety of the CP molecule, i.e., the terminal residues of sialic acids and a site, (formula; see text) located at a large distance from the chain terminus.

Benzophenone based fluorophore for selective detection of Sn2+ ion: Experimental and theoretical study.

PubMed

Jadhav, Amol G; Shinde, Suvidha S; Lanke, Sandip K; Sekar, Nagaiyan

2017-03-05

Synthesis of novel benzophenone-based chemosensor is presented for the selective sensing of Sn 2+ ion. Screening of competitive metal ions was performed by competitive experiments. The specific cation recognition ability of chemosensor towards Sn 2+ was investigated by experimental (UV-visible, fluorescence spectroscopy, 1 H NMR, 13 C NMR, FTIR and HRMS) methods and further supported by Density Functional Theory study. The stoichiometric binding ratio and binding constant (K a ) for complex is found to be 1:1 and 1.50×10 4 , respectively. The detection limit of Sn 2+ towards chemosensor was found to be 0.3898ppb. Specific selectivity and superiority of chemosensor over another recently reported chemosensor is presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

PubMed

Saito, Kazuki; Nakato, Mamiko; Mizuguchi, Takaaki; Wada, Shinji; Uchimura, Hiromasa; Kataoka, Hiroshi; Yokoyama, Shigeyuki; Hirota, Hiroshi; Kiso, Yoshiaki

2014-03-01

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, β-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 μM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Crystal-contact engineering to obtain a crystal form of the Kelch domain of human Keap1 suitable for ligand-soaking experiments.

PubMed

Hörer, Stefan; Reinert, Dirk; Ostmann, Katja; Hoevels, Yvette; Nar, Herbert

2013-06-01

Keap1 is a substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex and plays an important role in the cellular response to oxidative stress. It binds Nrf2 with its Kelch domain and thus triggers the ubiquitinylation and degradation of Nrf2. Oxidative stress prevents the degradation of Nrf2 and leads to the activation of cytoprotective genes. Therefore, Keap1 is an attractive drug target in inflammatory diseases. The support of a medicinal chemistry effort by structural research requires a robust crystallization system in which the crystals are preferably suited for performing soaking experiments. This facilitates the generation of protein-ligand complexes in a routine and high-throughput manner. The structure of human Keap1 has been described previously. In this crystal form, however, the binding site for Nrf2 was blocked by a crystal contact. This interaction was analysed and mutations were introduced to disrupt this crystal contact. One double mutation (E540A/E542A) crystallized in a new crystal form in which the binding site for Nrf2 was not blocked and was accessible to small-molecule ligands. The crystal structures of the apo form of the mutated Keap1 Kelch domain (1.98 Å resolution) and of the complex with an Nrf2-derived peptide obtained by soaking (2.20 Å resolution) are reported.

A survey of the year 2007 literature on applications of isothermal titration calorimetry.

PubMed

Bjelić, Sasa; Jelesarov, Ilian

2008-01-01

Elucidation of the energetic principles of binding affinity and specificity is a central task in many branches of current sciences: biology, medicine, pharmacology, chemistry, material sciences, etc. In biomedical research, integral approaches combining structural information with in-solution biophysical data have proved to be a powerful way toward understanding the physical basis of vital cellular phenomena. Isothermal titration calorimetry (ITC) is a valuable experimental tool facilitating quantification of the thermodynamic parameters that characterize recognition processes involving biomacromolecules. The method provides access to all relevant thermodynamic information by performing a few experiments. In particular, ITC experiments allow to by-pass tedious and (rarely precise) procedures aimed at determining the changes in enthalpy and entropy upon binding by van't Hoff analysis. Notwithstanding limitations, ITC has now the reputation of being the "gold standard" and ITC data are widely used to validate theoretical predictions of thermodynamic parameters, as well as to benchmark the results of novel binding assays. In this paper, we discuss several publications from 2007 reporting ITC results. The focus is on applications in biologically oriented fields. We do not intend a comprehensive coverage of all newly accumulated information. Rather, we emphasize work which has captured our attention with originality and far-reaching analysis, or else has provided ideas for expanding the potential of the method. Copyright (c) 2008 John Wiley & Sons, Ltd.

Task demands determine comparison strategy in whole probe change detection.

PubMed

Udale, Rob; Farrell, Simon; Kent, Chris

2018-05-01

Detecting a change in our visual world requires a process that compares the external environment (test display) with the contents of memory (study display). We addressed the question of whether people strategically adapt the comparison process in response to different decision loads. Study displays of 3 colored items were presented, followed by 'whole-display' probes containing 3 colored shapes. Participants were asked to decide whether any probed items contained a new feature. In Experiments 1-4, irrelevant changes to the probed item's locations or feature bindings influenced memory performance, suggesting that participants employed a comparison process that relied on spatial locations. This finding occurred irrespective of whether participants were asked to decide about the whole display, or only a single cued item within the display. In Experiment 5, when the base-rate of changes in the nonprobed items increased (increasing the incentive to use the cue effectively), participants were not influenced by irrelevant changes in location or feature bindings. In addition, we observed individual differences in the use of spatial cues. These results suggest that participants can flexibly switch between spatial and nonspatial comparison strategies, depending on interactions between individual differences and task demand factors. These findings have implications for models of visual working memory that assume that the comparison between study and test obligatorily relies on accessing visual features via their binding to location. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Muscarinic receptor binding increases in anterior thalamus and cingulate cortex during discriminative avoidance learning

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

Vogt, B.A.; Gabriel, M.; Vogt, L.J.

Training-induced neuronal activity develops in the mammalian limbic system during discriminative avoidance conditioning. This study explores behaviorally relevant changes in muscarinic ACh receptor binding in 52 rabbits that were trained to one of five stages of conditioned response acquisition. Sixteen naive and 10 animals yoked to criterion performance served as control cases. Upon reaching a particular stage of training, the brains were removed and autoradiographically assayed for 3H-oxotremorine-M binding with 50 nM pirenzepine (OxO-M/PZ) or for 3H-pirenzepine binding in nine limbic thalamic nuclei and cingulate cortex. Specific OxO-M/PZ binding increased in the parvocellular division of the anterodorsal nucleus early inmore » training when the animals were first exposed to pairing of the conditional and unconditional stimuli. Elevated binding in this nucleus was maintained throughout subsequent training. In the parvocellular division of the anteroventral nucleus (AVp), OxO-M/PZ binding progressively increased throughout training, reached a peak at the criterion stage of performance, and returned to control values during extinction sessions. Peak OxO-M/PZ binding in AVp was significantly elevated over that for cases yoked to criterion performance. In the magnocellular division of the anteroventral nucleus (AVm), OxO-M/PZ binding was elevated only during criterion performance of the task, and it was unaltered in any other limbic thalamic nuclei. Specific OxO-M/PZ binding was also elevated in most layers in rostral area 29c when subjects first performed a significant behavioral discrimination. Training-induced alterations in OxO-M/PZ binding in AVp and layer Ia of area 29c were similar and highly correlated.« less

In-situ Geochronology on the Mars 2020 Rover with KArLE (The Potassium-Argon Laser Experiment)

NASA Technical Reports Server (NTRS)

Cohen, Barbara A.; Li, Z. -H.; Miller, J. S.; Devismes, D.; Swindle, T. D.; Schwenzer, S. P.; Kelley, S. P.; Zacny, K. A.; Roark, S. E.; Hardaway, L. R.;

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens.

PubMed

Bel, Yolanda; Sheets, Joel J; Tan, Sek Yee; Narva, Kenneth E; Escriche, Baltasar

2017-06-01

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens ) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125 -iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. Copyright © 2017 Bel et al.

Energetics of dendrimer binding to HIV-1 gp120-CD4 complex and mechanismic aspects of its role as an entry-inhibitor

NASA Astrophysics Data System (ADS)

Saurabh, Suman; Sahoo, Anil Kumar; Maiti, Prabal K.

2016-10-01

Experiments and computational studies have established that de-protonated dendrimers (SPL7013 and PAMAM) act as entry-inhibitors of HIV. SPL7013 based Vivagel is currently under clinical development. The dendrimer binds to gp120 in the gp120-CD4 complex, destabilizes it by breaking key contacts between gp120 and CD4 and prevents viral entry into target cells. In this work, we provide molecular details and energetics of the formation of the SPL7013-gp120-CD4 ternary complex and decipher modes of action of the dendrimer in preventing viral entry. It is also known from experiments that the dendrimer binds weakly to gp120 that is not bound to CD4. It binds even more weakly to the CD4-binding region of gp120 and thus cannot directly block gp120-CD4 complexation. In this work, we examine the feasibility of dendrimer binding to the gp120-binding region of CD4 and directly blocking gp120-CD4 complex formation. We find that the process of the dendrimer binding to CD4 can compete with gp120-CD4 binding due to comparable free energy change for the two processes, thus creating a possibility for the dendrimer to directly block gp120-CD4 complexation by binding to the gp120-binding region of CD4.

Growth regulation of the mammalian ocular lens by vitreous humor.

PubMed

Banerjee, A; Parafina, J; Bagchi, M

1992-05-01

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95 degrees C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor-receptor binding is essential for the growth inhibitory effect.

Achromatic synesthesias - a functional magnetic resonance imaging study.

PubMed

Melero, H; Ríos-Lago, M; Peña-Melián, A; Álvarez-Linera, J

2014-09-01

Grapheme-color synesthetes experience consistent, automatic and idiosyncratic colors associated with specific letters and numbers. Frequently, these specific associations exhibit achromatic synesthetic qualities (e.g. white, black or gray). In this study, we have investigated for the first time the neural basis of achromatic synesthesias, their relationship to chromatic synesthesias and the achromatic congruency effect in order to understand not only synesthetic color but also other components of the synesthetic experience. To achieve this aim, functional magnetic resonance imaging experiments were performed in a group of associator grapheme-color synesthetes and matched controls who were stimulated with real chromatic and achromatic stimuli (Mondrians), and with letters and numbers that elicited different types of grapheme-color synesthesias (i.e. chromatic and achromatic inducers which elicited chromatic but also achromatic synesthesias, as well as congruent and incongruent ones). The information derived from the analysis of Mondrians and chromatic/achromatic synesthesias suggests that real and synesthetic colors/achromaticity do not fully share neural mechanisms. The whole-brain analysis of BOLD signals in response to the complete set of synesthetic inducers revealed that the functional peculiarities of the synesthetic brain are distributed, and reflect different components of the synesthetic experience: a perceptual component, an (attentional) feature binding component, and an emotional component. Additionally, the inclusion of achromatic experiences has provided new evidence in favor of the emotional binding theory, a line of interpretation which constitutes a bridge between grapheme-color synesthesia and other developmental modalities of the phenomenon. Copyright © 2014 Elsevier Inc. All rights reserved.

Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations

NASA Astrophysics Data System (ADS)

Wolff, Philippe; Da Veiga, Cyrielle; Ennifar, Eric; Bec, Guillaume; Guichard, Gilles; Burnouf, Dominique; Dumas, Philippe

2017-02-01

We studied by native ESI-MS the binding of various DNA-polymerase-derived peptides onto DNA-polymerase processivity rings from Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. These homodimeric rings present two equivalent specific binding sites, which leads to successive formation during a titration experiment of singly- and doubly occupied rings. By using the ESI-MS free-ring spectrum as a ruler, we derived by robust linear regression the fractions of the different ring species at each step of a titration experiment. These results led to accurate Kd values (from 0.03 to 0.5 μM) along with the probability of peptide loss due to gas phase dissociation (GPD). We show that this good quality is due to the increased information content of a titration experiment with a homodimer. Isothermal titration calorimetry (ITC) led with the same binding model to Kd(ITC) values systematically higher than their ESI-MS counterparts and, often, to poor fit of the ITC curves. A processing with two competing modes of binding on the same site requiring determination of two (Kd, ΔH) pairs greatly improved the fits and yielded a second Kd(ITC) close to Kd(ESI-MS). The striking features are: (1) ITC detected a minor binding mode ( 20%) of `low-affinity' that did not appear with ESI-MS; (2) the simplest processing of ITC data with only one (Kd, ΔH) pair led wrongly to the Kd of the low-affinity binding mode but to the ΔH of the high-affinity binding mode. Analogous misleading results might well exist in published data based on ITC experiments.

Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations.

PubMed

Wolff, Philippe; Da Veiga, Cyrielle; Ennifar, Eric; Bec, Guillaume; Guichard, Gilles; Burnouf, Dominique; Dumas, Philippe

2017-02-01

We studied by native ESI-MS the binding of various DNA-polymerase-derived peptides onto DNA-polymerase processivity rings from Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. These homodimeric rings present two equivalent specific binding sites, which leads to successive formation during a titration experiment of singly- and doubly occupied rings. By using the ESI-MS free-ring spectrum as a ruler, we derived by robust linear regression the fractions of the different ring species at each step of a titration experiment. These results led to accurate K d values (from 0.03 to 0.5 μM) along with the probability of peptide loss due to gas phase dissociation (GPD). We show that this good quality is due to the increased information content of a titration experiment with a homodimer. Isothermal titration calorimetry (ITC) led with the same binding model to K d (ITC) values systematically higher than their ESI-MS counterparts and, often, to poor fit of the ITC curves. A processing with two competing modes of binding on the same site requiring determination of two (K d , ΔH) pairs greatly improved the fits and yielded a second K d (ITC) close to K d (ESI-MS). The striking features are: (1) ITC detected a minor binding mode (~20%) of 'low-affinity' that did not appear with ESI-MS; (2) the simplest processing of ITC data with only one (K d , ΔH) pair led wrongly to the Kd of the low-affinity binding mode but to the ΔH of the high-affinity binding mode. Analogous misleading results might well exist in published data based on ITC experiments. Graphical Abstract ᅟ.

Effects of chronic delta-9-tetrahydrocannabinol (THC) administration on neurotransmitter concentrations and receptor binding in the rat brain

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

Ali, S.F.; Newport, G.D.; Scallet, A.C.

THC is the major psychoactive constituent of marijuana and is also known as an hallucinogenic compound. Numerous reports have shown that large doses of THC produce significant alterations in various neurotransmitter systems. The present study was designed to determine whether chronic exposure to THC produces significant alterations in selected neurotransmitter systems (dopamine, serotonin, acetylcholine, GABAergic, benzodiazepine, and opiate) in the rat brain. In Experiment 1, male Sprague-Dawley rats were gavaged with vehicle, 10 or 20 mg THC/kg body weight daily, 5 days/week for 90 days. Animals were killed either 24 hours or two months after the last dose. Brains weremore » dissected into different regions for neurochemical analyses. Two months after the cessation of chronic administration, there was a significant decrease in GABA receptor binding in the hippocampus of animals in the high dose group. However, no other significant changes were found in neurotransmitter receptor binding characteristics in the hippocampus or in neurotransmitter concentrations in the caudate nucleus, hypothalamus or septum after chronic THC administration. In an attempt to replicate the GABA receptor binding changes and also to determine the (35S)TBPS binding in hippocampus, we designed Experiment 2. In this experiment, we dosed the animals by gavage with 0, 5, 10 or 20 mg THC/kg daily, 5 days/week or with 20 mg THC/kg Monday through Thursday and 60 mg/kg on Friday for 90 days. Results from this experiment failed to replicate the dose-dependent effect of THC on GABA receptor binding in hippocampus. Modulation of (35S)TBPS binding by GABA or 3 alpha-OH-DHP or inhibition by cold TBPS in frontal cortex did not show any significant dose-related effects.« less

Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

ERIC Educational Resources Information Center

Ingersoll, Christine M.; Strollo, Christen M.

2007-01-01

The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

A global optimization algorithm for protein surface alignment

PubMed Central

2010-01-01

Background A relevant problem in drug design is the comparison and recognition of protein binding sites. Binding sites recognition is generally based on geometry often combined with physico-chemical properties of the site since the conformation, size and chemical composition of the protein surface are all relevant for the interaction with a specific ligand. Several matching strategies have been designed for the recognition of protein-ligand binding sites and of protein-protein interfaces but the problem cannot be considered solved. Results In this paper we propose a new method for local structural alignment of protein surfaces based on continuous global optimization techniques. Given the three-dimensional structures of two proteins, the method finds the isometric transformation (rotation plus translation) that best superimposes active regions of two structures. We draw our inspiration from the well-known Iterative Closest Point (ICP) method for three-dimensional (3D) shapes registration. Our main contribution is in the adoption of a controlled random search as a more efficient global optimization approach along with a new dissimilarity measure. The reported computational experience and comparison show viability of the proposed approach. Conclusions Our method performs well to detect similarity in binding sites when this in fact exists. In the future we plan to do a more comprehensive evaluation of the method by considering large datasets of non-redundant proteins and applying a clustering technique to the results of all comparisons to classify binding sites. PMID:20920230

Elucidation of intermolecular interaction of bovine serum albumin with Fenhexamid: A biophysical prospect.

PubMed

Shi, Jie-Hua; Lou, Yan-Yue; Zhou, Kai-Li; Pan, Dong-Qi

2018-03-01

Fenhexamid, as a hydroxyanilide, is widely applied to control Botrytis cinerea for protecting crops and fruits. But it could adversely affect human and animals health due to accumulation of residues in food production. Here, the affinity characteristics of fenhexamid on bovine serum albumin (BSA) was studied via a series of spectroscopic methods such as steady-state fluorescence spectroscopy, ultraviolet spectroscopy (UV), synchronous fluorescence spectroscopy (SFS), 3D fluorescence spectroscopy, and fourier transform infrared spectroscopy (FT-IR). The experimental results illustrated that the fluorescence quenching mechanism of BSA induced by fenhexamid was a static quenching. The binding constant (K b ) of fenhexamid with BSA was 2.399 × 10 4  M -1 at 298 K and the combination ratio was about 1:1. The competitive experiment demonstrated that fenhexamid was binding on the BSA at site II (subdomain IIIA), which was confirmed by the molecular docking studies. The negative values of thermodynamic parameter (ΔH 0 , ΔS 0 and ΔG 0 ) revealed that the reaction of fenhexamid with BSA could proceed spontaneously, the van der Waals force and hydrogen bonding interaction conducted the main effect, and the binding process was enthalpy-driven. What's more, the 8-Anilino-1-naphthalenesulfonate (ANS) and sucrose binding studies were also performed and further verified the binding force between BSA and fenhexamid. Copyright © 2018 Elsevier B.V. All rights reserved.

Evidence for glucocorticoid receptor binding to a site(s) in a remote region of the 5' flanking sequences of the human proopiomelanocortin gene

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

Tully, D.B.; Hillman, D.; Herbert, E.

1986-05-01

Glucocorticoids negatively regulate expression of the human proopiomelanocortin (POMC) gene. It has been postulated that this effect may be modulated by a direct interaction of the glucocorticoid receptor (GR) with DNA in the vicinity of the POMC promoter. In order to investigate interactions of GR with POMC DNA, DNA-cellulose competitive binding assays have been performed using isolated fragments of cloned POMC DNA to compete with calf thymus DNA-cellulose for binding of triamcinolone acetonide affinity-labelled GR prepared from HeLa S/sub 3/ cells. In these assays, two fragments isolated from the 5' flanking sequences of POMC DNA (Fragment 3,-1765 to -677 andmore » Fragment 4, -676 to +125 with respect to the mRNA cap site) have competed favorably, with Fragment 3 consistently competing more strongly than Fragment 4. Additional studies have been conducted utilizing a newly developed South-western Blot procedure in which specific /sup 32/P-labelled DNA fragments are allowed to bind to dexamethasone mesylate labelled GR immobilized on nitrocellulose filters. Results from these studies have also shown preferential binding by POMC DNA fragments 3 and 4. DNA footprinting and gene transfer experiments are now being conducted to further characterize the nature of GR interaction with POMC DNA.« less

An ab initio study of Fe(CO)n, n = 1,5, and Cr(CO)6

NASA Technical Reports Server (NTRS)

Barnes, Leslie A.; Rosi, Marzio; Bauschlicher, Charles W., Jr.

1991-01-01

Ab initio calculations have been performed for Cr(CO)6 and Fe(CO)n, n = 1,5. Basis sets of better than double zeta quality are used, and correlation is included using the modified coupler-pair functional method. The computed geometries and force constants are in reasonable agreement with experiment. The sequential bond dissociation energies of CO from Fe(CO)5 are estimated to be: 39, 31, 25, 22, and greater than 5 kcal/mol. It is noted that the first bond dissociation energy is relative to the singlet ground state of Fe(CO)5 and the lowest singlet state of Fe(CO)4, whereas the second is relative to the ground triplet states of Fe(CO)4 and Fe(CO)3. In addition, the binding energy for Fe-CO would be modified to 18 kcal/mol if dissociation occurred to the Fe(5F) excited state asymptote. The CO binding energies for Fe and Cr are found to be in poorer agreement with experiment than those found in a previous study on Ni(CO)4. The origins of this difference are discussed.

A bacteria antibiotic system in space (23-F ANTIBIO)

NASA Technical Reports Server (NTRS)

Tixador, Rene; Gasset, G.; Eche, B.; Moatti, N.; Lapchine, L.; Woldringh, C.; Toorop, P.; Moatti, J. P.; Delmotte, F.; Tap, G.

1995-01-01

In order to evaluate the effects of weightlessness and cosmic radiations on the bacteria resistance to antibiotics, the Antibio 23F experiment was undertaken onboard Discovery during the 1st International Microgravity Laboratory (IML-1) mission. The effects of various antibiotic concentrations (dihydrostreptomycin) on Escherichia coli growth and cell division behavior were studied. The antibiotic binding was investigated using a radioactive tracer (tritium). The results showed that microgravity did not affect E. coli cells in regards the growth and the cell division. The antibiotic added to the culture medium induced an inhibition of the cultures both in the flight and ground controls. However, the antibiotic was less efficient in flight. The behavior of bacteria was modified, and the exponential growth rate was increased in flight. The incorporation of radioactive antibiotics in flight was comparatively different to ground incorporation, which indicated some perturbations in antibiotic binding. The experiments performed in the 1 g centrifuge did not show any difference in the cultures developed on the static rack, and could support a radiative effect of cosmic radiation to explain the results.

Episodic Memory and Future Thinking During Early Childhood: Linking the Past and Future

PubMed Central

Cuevas, Kimberly; Rajan, Vinaya; Morasch, Katherine C.; Bell, Martha Ann

2015-01-01

Despite extensive examination of episodic memory and future thinking development, little is known about the concurrent emergence of these capacities during early childhood. In Experiment 1, 3-year-olds participated in an episodic memory hiding task [“what, when, where” (WWW) components] with an episodic future thinking component. In Experiment 2, a group of 4-year-olds (including children from Experiment 1) participated in the same task (different objects and locations), providing the first longitudinal investigation of episodic memory and future thinking. Although children exhibited age-related improvements in recall, recognition, and binding of the WWW episodic memory components, there were no age-related changes in episodic future thinking. At both ages, WWW episodic memory performance was higher than future thinking performance, and episodic future thinking and WWW memory components were unrelated. These findings suggest that the WWW components of episodic memory are potentially less fragile than the future components when assessed in a cognitively demanding task. PMID:25864990

Episodic memory and future thinking during early childhood: Linking the past and future.

PubMed

Cuevas, Kimberly; Rajan, Vinaya; Morasch, Katherine C; Bell, Martha Ann

2015-07-01

Despite extensive examination of episodic memory and future thinking development, little is known about the concurrent emergence of these capacities during early childhood. In Experiment 1, 3-year-olds participated in an episodic memory hiding task ("what, when, where" [WWW] components) with an episodic future thinking component. In Experiment 2, a group of 4-year-olds (including children from Experiment 1) participated in the same task (different objects and locations), providing the first longitudinal investigation of episodic memory and future thinking. Although children exhibited age-related improvements in recall, recognition, and binding of the WWW episodic memory components, there were no age-related changes in episodic future thinking. At both ages, WWW episodic memory performance was higher than future thinking performance, and episodic future thinking and WWW memory components were unrelated. These findings suggest that the WWW components of episodic memory are potentially less fragile than the future components when assessed in a cognitively demanding task. © 2015 Wiley Periodicals, Inc.

Radioiodination and preclinical evaluation of 4-benzyl-1-(3-[125I]-iodobenzylsulfonyl)piperidine as a breast tumor imaging tracer in mouse.

PubMed

Sadeghzadeh, Masoud; Alirezapour, Behrouz; Charkhlooie, Ghorban Ali; Baghery, Maryam Keshavarz; Khorouti, Amir

2017-05-01

4-Benzyl-1-(3-iodobenzylsulfonyl)piperidine, 4-B-IBSP, has shown high-binding affinity to both sigma (σ) receptors in our previous work. In current study, radiolabeling and preclinical evaluation of 4-benzyl-1-(3-[ 125 I]-iodobenzylsulfonyl)piperidine, 4-B-[ 125 I]IBSP, in human ductal breast carcinoma (T47D) cells and in breast adenocarcinoma-bearing BALB/c mice are described. Radioiodination of this new σ ligand was performed by a palladium-catalyzed stannylation approach followed by oxidative iododestannylation reaction using Iodo-Gen. Competition-binding assays for binding of 4-B-[ 125 I]IBSP to guinea pig brain membranes and to T47D cells were performed with known σ ligands. The selectivity and binding characteristics (B max and K d ) were analyzed. In vitro stability and in vivo blood metabolism studies were also evaluated. Moreover, biodistribution studies were performed in normal and into the tumor-bearing mice at interval time points post-injection (p.i.). Both in vitro and in vivo blockade experiments were done in the presence of the σ receptors blocking agents. Radioiodinated ligand was obtained in high yield and high specific activity. The σ inhibition constants (K i , nM) for 4-(3-iodobenzyl)-1-(benzylsulfonyl)piperazine (4-IBBSPz), (+)-pentazocine, haloperidol, DTG, and 4-B-IBSP were 1.37 ± 0.19, 3.90 ± 0.77, 2.69 ± 0.33, 30.62 ± 2.01, and 0.61 ± 0.05, respectively. 4-B-[ 125 I]IBSP bound to σ receptor sites preferably to very high-affinity binding sites on T47D cells. The radioligand showed acceptable in vitro and in vivo stabilities in the blood pool. However, in vivo biodistribution studies in normal Swiss albino mice revealed fast clearance of 4-B-[ 125 I]IBSP from blood and the other normal organs. Biodistribution experiments of 4-B-[ 125 I]IBSP in breast adenocarcinoma tumor-bearing BALB/c mice showed a relatively high tumor uptake at 30 min p.i. (4.13 ± 0.95) that reaches to 1.57 ± 0.24 even after 240 min p.i. A pre-injection of 4-B-IBSP and haloperidol with 4-B-[ 125 I]IBSP resulted in 36-57% decrease in activity in the tumor, liver, and brain at 60 min p.i. The high affinity of 4-B-[ 125 I]IBSP to σ receptor-binding sites, its relatively high uptake, and preferential retention in the tumor as well as an increasing trend observed in the tumor to blood and in the tumor to muscle ratios suggests that an iodine-123 labeled counterpart, 4-B-[ 123 I]IBSP, would be a promising σ radioligand for pursuing further studies to assess its potential for breast tumors imaging with SPECT.

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

PubMed Central

Arnaut, Vera; Langecker, Martin; Simmel, Friedrich C.

2013-01-01

The stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified. PMID:24010663

A revised design for microarray experiments to account for experimental noise and uncertainty of probe response.

PubMed

Pozhitkov, Alex E; Noble, Peter A; Bryk, Jarosław; Tautz, Diethard

2014-01-01

Although microarrays are analysis tools in biomedical research, they are known to yield noisy output that usually requires experimental confirmation. To tackle this problem, many studies have developed rules for optimizing probe design and devised complex statistical tools to analyze the output. However, less emphasis has been placed on systematically identifying the noise component as part of the experimental procedure. One source of noise is the variance in probe binding, which can be assessed by replicating array probes. The second source is poor probe performance, which can be assessed by calibrating the array based on a dilution series of target molecules. Using model experiments for copy number variation and gene expression measurements, we investigate here a revised design for microarray experiments that addresses both of these sources of variance. Two custom arrays were used to evaluate the revised design: one based on 25 mer probes from an Affymetrix design and the other based on 60 mer probes from an Agilent design. To assess experimental variance in probe binding, all probes were replicated ten times. To assess probe performance, the probes were calibrated using a dilution series of target molecules and the signal response was fitted to an adsorption model. We found that significant variance of the signal could be controlled by averaging across probes and removing probes that are nonresponsive or poorly responsive in the calibration experiment. Taking this into account, one can obtain a more reliable signal with the added option of obtaining absolute rather than relative measurements. The assessment of technical variance within the experiments, combined with the calibration of probes allows to remove poorly responding probes and yields more reliable signals for the remaining ones. Once an array is properly calibrated, absolute quantification of signals becomes straight forward, alleviating the need for normalization and reference hybridizations.

The Effect of Hydraulic Loading Rate and Influent Source on the Binding Capacity of Phosphorus Filters

PubMed Central

Herrmann, Inga; Jourak, Amir; Hedström, Annelie; Lundström, T. Staffan; Viklander, Maria

2013-01-01

Sorption by active filter media can be a convenient option for phosphorus (P) removal and recovery from wastewater for on-site treatment systems. There is a need for a robust laboratory method for the investigation of filter materials to enable a reliable estimation of their longevity. The objectives of this study were to (1) investigate and (2) quantify the effect of hydraulic loading rate and influent source (secondary wastewater and synthetic phosphate solution) on P binding capacity determined in laboratory column tests and (3) to study how much time is needed for the P to react with the filter material (reaction time). To study the effects of these factors, a 22 factorial experiment with 11 filter columns was performed. The reaction time was studied in a batch experiment. Both factors significantly (α = 0.05) affected the P binding capacity negatively, but the interaction of the two factors was not significant. Increasing the loading rate from 100 to 1200 L m−2 d−1 decreased P binding capacity from 1.152 to 0.070 g kg−1 for wastewater filters and from 1.382 to 0.300 g kg−1 for phosphate solution filters. At a loading rate of 100 L m−2 d−1, the average P binding capacity of wastewater filters was 1.152 g kg−1 as opposed to 1.382 g kg−1 for phosphate solution filters. Therefore, influent source or hydraulic loading rate should be carefully controlled in the laboratory. When phosphate solution and wastewater were used, the reaction times for the filters to remove P were determined to be 5 and 15 minutes, respectively, suggesting that a short residence time is required. However, breakthrough in this study occurred unexpectedly quickly, implying that more time is needed for the P that has reacted to be physically retained in the filter. PMID:23936313

Revisiting point FRAP to quantitatively characterize anomalous diffusion in live cells.

PubMed

Daddysman, Matthew K; Fecko, Christopher J

2013-02-07

Fluorescence recovery after photobleaching (FRAP) is widely used to interrogate diffusion and binding of proteins in live cells. Herein, we apply two-photon excited FRAP with a diffraction limited bleaching and observation volume to study anomalous diffusion of unconjugated green fluorescence protein (GFP) in vitro and in cells. Experiments performed on dilute solutions of GFP reveal that reversible fluorophore bleaching can be mistakenly interpreted as anomalous diffusion. We derive a reaction-diffusion FRAP model that includes reversible photobleaching, and demonstrate that it properly accounts for these photophysics. We then apply this model to investigate the diffusion of GFP in HeLa cells and polytene cells of Drosophila larval salivary glands. GFP exhibits anomalous diffusion in the cytoplasm of both cell types and in HeLa nuclei. Polytene nuclei contain optically resolvable chromosomes, permitting FRAP experiments that focus separately on chromosomal or interchrosomal regions. We find that GFP exhibits anomalous diffusion in chromosomal regions but diffuses normally in regions devoid of chromatin. This observation indicates that obstructed transport through chromatin and not crowding by macromolecules is a source of anomalous diffusion in polytene nuclei. This behavior is likely true in other cells, so it will be important to account for this type of transport physics and for reversible photobleaching to properly interpret future FRAP experiments on DNA-binding proteins.

Imaging label-free biosensor with microfluidic system

NASA Astrophysics Data System (ADS)

Jahns, S.; Glorius, P.; Hansen, M.; Nazirizadeh, Y.; Gerken, M.

2015-06-01

We present a microfluidic system suitable for parallel label-free detection of several biomarkers utilizing a compact imaging measurement system. The microfluidic system contains a filter unit to separate the plasma from human blood and a functionalized, photonic crystal slab sensor chip. The nanostructure of the photonic crystal slab sensor chip is fabricated by nanoimprint lithography of a period grating surface into a photoresist and subsequent deposition of a TiO2 layer. Photonic crystal slabs are slab waveguides supporting quasi-guided modes coupling to far-field radiation, which are sensitive to refractive index changes due to biomarker binding on the functionalized surface. In our imaging read-out system the resulting resonance shift of the quasi-guided mode in the transmission spectrum is converted into an intensity change detectable with a simple camera. By continuously taking photographs of the sensor surface local intensity changes are observed revealing the binding kinetics of the biomarker to its specific target. Data from two distinct measurement fields are used for evaluation. For testing the sensor chip, 1 μM biotin as well as 1 μM recombinant human CD40 ligand were immobilized in spotsvia amin coupling to the sensor surface. Each binding experiment was performed with 250 nM streptavidin and 90 nM CD40 ligand antibody dissolved in phosphate buffered saline. In the next test series, a functionalized sensor chip was bonded onto a 15 mm x 15 mm opening of the 75 mm x 25 mm x 2 mm microfluidic system. We demonstrate the functionality of the microfluidic system for filtering human blood such that only blood plasma was transported to the sensor chip. The results of first binding experiments in buffer with this test chip will be presented.

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

PubMed

Rastaldi, M P; Candiano, G; Musante, L; Bruschi, M; Armelloni, S; Rimoldi, L; Tardanico, R; Sanna-Cherchi, S; Cherchi, S Sanna; Ferrario, F; Montinaro, V; Haupt, R; Parodi, S; Carnevali, M L; Allegri, L; Camussi, G; Gesualdo, L; Scolari, F; Ghiggeri, G M

2006-08-01

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

Agonist-dependent consequences of proline to alanine substitution in the transmembrane helices of the calcitonin receptor

PubMed Central

Bailey, R J; Hay, D L

2007-01-01

Background and purpose: Transmembrane proline (P) residues in family A G protein-coupled receptors (GPCRs) form functionally important kinks in their helices. These residues are little studied in family B GPCRs but experiments with the VPAC1 receptor and calcitonin receptor-like receptor (CL) show parallels with family A receptors. We sought to determine the function of these residues in the insert negative form of the human calcitonin receptor, a close relative of CL. Experimental approach: Proline residues within the transmembrane domains of the calcitonin receptor (P246, P249, P280, P326, P336) were individually mutated to alanine (A) using site-directed mutagenesis. Receptors were transiently transfected into Cos-7 cells using polyethylenimine and salmon and human calcitonin-induced cAMP responses measured. Salmon and human calcitonin competition binding experiments were also performed and receptor cell-surface expression assessed by whole cell ELISA. Key results: P246A, P249A and P280A were wild-type in terms of human calcitonin-induced cAMP activation. P326A and P336A had reduced function (165 and 12-fold, respectively). In membranes, human calcitonin binding was not detectable for any mutant receptor but in whole cells, binding was detected for all mutants apart from P326A. Salmon calcitonin activated mutant and wild-type receptors equally, although Bmax values were reduced for all mutants apart from P326A. Conclusions and Implications: P326 and P336 are important for the function of human calcitonin receptors and are likely to be involved in generating receptor conformations appropriate for agonist binding and receptor activation. However, agonist-specific effects were observed , implying distinct conformations of the human calcitonin receptor. PMID:17486143

Pretargeting of carcinoembryonic antigen-expressing cancers with a trivalent bispecific fusion protein produced in myeloma cells.

PubMed

Rossi, Edmund A; Chang, Chien-Hsing; Losman, Michele J; Sharkey, Robert M; Karacay, Habibe; McBride, William; Cardillo, Thomas M; Hansen, Hans J; Qu, Zhengxing; Horak, Ivan D; Goldenberg, David M

2005-10-01

To characterize a novel trivalent bispecific fusion protein and evaluate its potential utility for pretargeted delivery of radionuclides to tumors. hBS14, a recombinant fusion protein that binds bispecifically to carcinoembryonic antigen (CEA) and the hapten, histamine-succinyl-glycine (HSG), was produced by transgenic myeloma cells and purified to near homogeneity in a single step using a novel HSG-based affinity chromatography system. Biochemical characterization included size-exclusion high-performance liquid chromatography (SE-HPLC), SDS-PAGE, and isoelectric focusing. Functional characterization was provided by BIAcore and SE-HPLC. The efficacy of hBS14 for tumor pretargeting was evaluated in CEA-expressing GW-39 human colon tumor-bearing nude mice using a bivalent HSG hapten (IMP-241) labeled with (111)In. Biochemical analysis showed that single-step affinity chromatography provided highly purified material. SE-HPLC shows a single protein peak consistent with the predicted molecular size of hBS14. SDS-PAGE analysis shows only two polypeptide bands, which are consistent with the calculated molecular weights of the hBS14 polypeptides. BIAcore showed the bispecific binding properties and suggested that hBS14 possesses two functional CEA-binding sites. This was supported by SE-HPLC immunoreactivity experiments. All of the data suggest that the structure of hBS14 is an 80 kDa heterodimer with one HSG and two CEA binding sites. Pretargeting experiments in the mouse model showed high uptake of radiopeptide in the tumor, with favorable tumor-to-nontumor ratios as early as 3 hours postinjection. The results indicate that hBS14 is an attractive candidate for use in a variety of pretargeting applications, particularly tumor therapy with radionuclides and drugs.

Investigation of differences in the binding affinities of two analogous ligands for untagged and tagged p38 kinase using thermodynamic integration MD simulation.

PubMed

Sun, Ying-Chieh; Hsu, Wen-Chi; Hsu, Chia-Jen; Chang, Chia-Ming; Cheng, Kai-Hsiang

2015-11-01

Thermodynamic integration (TI) molecular dynamics (MD) simulations for the binding of a pair of a reference ("ref") ligand and an analogous ("analog") ligand to either tagged (with six extra residues at the N-terminus) or untagged p38 kinase proteins were carried out in order to probe how the binding affinity is influenced by the presence or absence of the peptide tag in p38 kinase. This possible effect of protein length on the binding affinity of a ligand-which is seldom addressed in the literature-is important because, even when two labs claim to have performed experiments with the same protein, they may actually have studied variants of the same protein with different lengths because they applied different protein expression conditions/procedures. Thus, if we wanted to compare ligand binding affinities measured in the two labs, it would be necessary to account for any variation in ligand binding affinity with protein length. The pair of ligand-p38 kinase complexes examined in this work (pdb codes: 3d7z and 3lhj, respectively) were ideal for investigating this effect. The experimentally determined binding energy for the ref ligand with the untagged p38 kinase was -10.9 kcal mol(-1), while that for the analog ligand with the tagged p38 kinase was -11.9 kcal mol(-1). The present TI-MD simulation of the mutation of the ref ligand into the analog ligand while the ligand is bound to the untagged p38 kinase predicted that the binding affinity of the analog ligand is 2.0 kcal mol(-1) greater than that of the ref ligand. A similar simulation also indicated that the same was true for ligand binding to the tagged protein, but in this case the binding affinity for the analog ligand is 2.5 kcal mol(-1) larger than that for the ref ligand. These results therefore suggest that the presence of the peptide tag on p38 kinase increased the difference in the binding energies of the ligands by a small amount of 0.5 kcal mol(-1). This result supports the assumption that the presence of a peptide tag has only a minor effect on ΔG values. The error bars in the computed ΔG values were then estimated via confidence interval analysis and a time autocorrelation function for the quantity dV/dλ. The estimated correlation time was ~0.5 ps and the error bar in the ΔG values estimated using nanosecond-scale simulations was ±0.3 kcal mol(-1) at a confidence level of 95%. These predicted results can be verified in future experiments and should prove useful in subsequent similar studies. Graphical Abstract Thermodynamic cycles for binding of two analogous ligands with untagged and tagged p38 kinases and associated Gibbs free energy.

Differential Contributions of Selective Attention and Sensory Integration to Driving Performance in Healthy Aging and Alzheimer's Disease.

PubMed

Venkatesan, Umesh M; Festa, Elena K; Ott, Brian R; Heindel, William C

2018-05-01

Patients with Alzheimer's disease (AD) demonstrate deficits in cross-cortical feature binding distinct from age-related changes in selective attention. This may have consequences for driving performance given its demands on multisensory integration. We examined the relationship of visuospatial search and binding to driving in patients with early AD and elderly controls (EC). Participants (42 AD; 37 EC) completed search tasks requiring either luminance-motion (L-M) or color-motion (C-M) binding, analogs of within and across visual processing stream binding, respectively. Standardized road test (RIRT) and naturalistic driving data (CDAS) were collected alongside clinical screening measures. Patients performed worse than controls on most cognitive and driving indices. Visual search and clinical measures were differentially related to driving behavior across groups. L-M search and Trail Making Test (TMT-B) were associated with RIRT performance in controls, while C-M binding, TMT-B errors, and Clock Drawing correlated with CDAS performance in patients. After controlling for demographic and clinical predictors, L-M reaction time significantly predicted RIRT performance in controls. In patients, C-M binding made significant contributions to CDAS above and beyond demographic and clinical predictors. RIRT and C-M binding measures accounted for 51% of variance in CDAS performance in patients. Whereas selective attention is associated with driving behavior in EC, cross-cortical binding appears most sensitive to driving in AD. This latter relationship may emerge only in naturalistic settings, which better reflect patients' driving behavior. Visual integration may offer distinct insights into driving behavior, and thus has important implications for assessing driving competency in early AD. (JINS, 2018, 24, 486-497).

How to Illustrate Ligand-Protein Binding in a Class Experiment: An Elementary Fluorescent Assay.

ERIC Educational Resources Information Center

Marty, Alain; And Others

1986-01-01

Describes an experiment (taking approximately five hours) which illustrates the binding of a small molecule to a protein. By using an appropriate fluorescent ligand and a given protein, the fluorescent probe technique is applied to measure the number of bonding sites, and number of site classes, and their association constants. (JN)

Binding Objects to Locations: The Relationship between Object Files and Visual Working Memory

ERIC Educational Resources Information Center

Hollingworth, Andrew; Rasmussen, Ian P.

2010-01-01

The relationship between object files and visual working memory (VWM) was investigated in a new paradigm combining features of traditional VWM experiments (color change detection) and object-file experiments (memory for the properties of moving objects). Object-file theory was found to account for a key component of object-position binding in VWM:…

Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.

PubMed

Hedlund, P B; Carson, M J; Sutcliffe, J G; Thomas, E A

1999-12-01

Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

PubMed

Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

2016-07-19

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

Botulinum neurotoxin G binds synaptotagmin-II in a mode similar to that of serotype B: tyrosine 1186 and lysine 1191 cause its lower affinity.

PubMed

Willjes, Gesche; Mahrhold, Stefan; Strotmeier, Jasmin; Eichner, Timo; Rummel, Andreas; Binz, Thomas

2013-06-04

Botulinum neurotoxins (BoNTs) block neurotransmitter release by proteolyzing SNARE proteins in peripheral nerve terminals. Entry into neurons occurs subsequent to interaction with gangliosides and a synaptic vesicle protein. Isoforms I and II of synaptotagmin were shown to act as protein receptors for two of the seven BoNT serotypes, BoNT/B and BoNT/G, and for mosaic-type BoNT/DC. BoNT/B and BoNT/G exhibit a homologous binding site for synaptotagmin whose interacting part adopts helical structure upon binding to BoNT/B. Whereas the BoNT/B-synaptotagmin-II interaction has been elucidated in molecular detail, corresponding information about BoNT/G is lacking. Here we systematically mutated the synaptotagmin binding site in BoNT/G and performed a comparative binding analysis with mutants of the cell binding subunit of BoNT/B. The results suggest that synaptotagmin takes the same overall orientation in BoNT/B and BoNT/G governed by the strictly conserved central parts of the toxins' binding site. The surrounding nonconserved areas differently contribute to receptor binding. Reciprocal mutations Y1186W and L1191Y increased the level of binding of BoNT/G approximately to the level of BoNT/B affinity, suggesting a similar synaptotagmin-bound state. The effects of the mutations were confirmed by studying the activity of correspondingly mutated full-length BoNTs. On the basis of these data, molecular modeling experiments were employed to reveal an atomistic model of BoNT/G-synaptotagmin recognition. These data suggest a reduced length and/or a bend in the C-terminal part of the synaptotagmin helix that forms upon contact with BoNT/G as compared with BoNT/B and are in agreement with the data of the mutational analyses.

Lectin binding assays for in-process monitoring of sialylation in protein production.

PubMed

Xu, Weiduan; Chen, Jianmin; Yamasaki, Glenn; Murphy, John E; Mei, Baisong

2010-07-01

Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galbeta1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(beta1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galbeta1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galbeta1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.

The SAMPL4 host-guest blind prediction challenge: an overview.

PubMed

Muddana, Hari S; Fenley, Andrew T; Mobley, David L; Gilson, Michael K

2014-04-01

Prospective validation of methods for computing binding affinities can help assess their predictive power and thus set reasonable expectations for their performance in drug design applications. Supramolecular host-guest systems are excellent model systems for testing such affinity prediction methods, because their small size and limited conformational flexibility, relative to proteins, allows higher throughput and better numerical convergence. The SAMPL4 prediction challenge therefore included a series of host-guest systems, based on two hosts, cucurbit[7]uril and octa-acid. Binding affinities in aqueous solution were measured experimentally for a total of 23 guest molecules. Participants submitted 35 sets of computational predictions for these host-guest systems, based on methods ranging from simple docking, to extensive free energy simulations, to quantum mechanical calculations. Over half of the predictions provided better correlations with experiment than two simple null models, but most methods underperformed the null models in terms of root mean squared error and linear regression slope. Interestingly, the overall performance across all SAMPL4 submissions was similar to that for the prior SAMPL3 host-guest challenge, although the experimentalists took steps to simplify the current challenge. While some methods performed fairly consistently across both hosts, no single approach emerged as consistent top performer, and the nonsystematic nature of the various submissions made it impossible to draw definitive conclusions regarding the best choices of energy models or sampling algorithms. Salt effects emerged as an issue in the calculation of absolute binding affinities of cucurbit[7]uril-guest systems, but were not expected to affect the relative affinities significantly. Useful directions for future rounds of the challenge might involve encouraging participants to carry out some calculations that replicate each others' studies, and to systematically explore parameter options.

Computational scheme for pH-dependent binding free energy calculation with explicit solvent.

PubMed

Lee, Juyong; Miller, Benjamin T; Brooks, Bernard R

2016-01-01

We present a computational scheme to compute the pH-dependence of binding free energy with explicit solvent. Despite the importance of pH, the effect of pH has been generally neglected in binding free energy calculations because of a lack of accurate methods to model it. To address this limitation, we use a constant-pH methodology to obtain a true ensemble of multiple protonation states of a titratable system at a given pH and analyze the ensemble using the Bennett acceptance ratio (BAR) method. The constant pH method is based on the combination of enveloping distribution sampling (EDS) with the Hamiltonian replica exchange method (HREM), which yields an accurate semi-grand canonical ensemble of a titratable system. By considering the free energy change of constraining multiple protonation states to a single state or releasing a single protonation state to multiple states, the pH dependent binding free energy profile can be obtained. We perform benchmark simulations of a host-guest system: cucurbit[7]uril (CB[7]) and benzimidazole (BZ). BZ experiences a large pKa shift upon complex formation. The pH-dependent binding free energy profiles of the benchmark system are obtained with three different long-range interaction calculation schemes: a cutoff, the particle mesh Ewald (PME), and the isotropic periodic sum (IPS) method. Our scheme captures the pH-dependent behavior of binding free energy successfully. Absolute binding free energy values obtained with the PME and IPS methods are consistent, while cutoff method results are off by 2 kcal mol(-1) . We also discuss the characteristics of three long-range interaction calculation methods for constant-pH simulations. © 2015 The Protein Society.

Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues

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

Kruer-Zerhusen, Nathan; Alahuhta, Markus; Lunin, Vladimir V.

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The pointmore » mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.« less

Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues

DOE PAGES

Kruer-Zerhusen, Nathan; Alahuhta, Markus; Lunin, Vladimir V.; ...

2017-11-30

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The pointmore » mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.« less

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

PubMed Central

Bondarenko, Vasyl; Mowrey, David; Liu, Lu Tian; Xu, Yan; Tang, Pei

2012-01-01

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the 2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury, et. al. 2011). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions. PMID:23000369

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR.

PubMed

Bondarenko, Vasyl; Mowrey, David; Liu, Lu Tian; Xu, Yan; Tang, Pei

2013-02-01

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions. Copyright © 2012 Elsevier B.V. All rights reserved.

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5*

PubMed Central

Choudhury, Rawshan; McGovern, Amanda; Ridley, Caroline; Cain, Stuart A.; Baldwin, Andrew; Wang, Ming-Chuan; Guo, Chun; Mironov, Aleksandr; Drymoussi, Zoe; Trump, Dorothy; Shuttleworth, Adrian; Baldock, Clair; Kielty, Cay M.

2009-01-01

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils. PMID:19570982

Role of protons in the pump cycle of KdpFABC investigated by time-resolved kinetic experiments.

PubMed

Damnjanovic, Bojana; Apell, Hans-Jürgen

2014-05-20

The time-resolved kinetics of the KdpFABC complex solubilized in Aminoxide WS-35 was investigated by ATP concentration jump experiments. ATP was photoreleased from its inactive precursor, caged ATP, and charge movements in the membrane domain of the KdpFABC were detected by the electrochromic dye RH421. At low ATP concentrations, the ATP binding step became rate-limiting with an apparent, pH-independent ATP binding affinity of ~70 μM. At saturating ATP concentrations, the rate-limiting step is the conformational transition (E1-P → P-E2) with a rate constant of ~1.7 s(-1) at 20 °C that was independent of K(+) concentration. This observation together with the detected fluorescence decrease indicates that K(+) (or another positive ion) is bound in the membrane domain after enzyme phosphorylation and the conformational transition to the P-E2 state. pH dependence experiments revealed different roles of H(+) in the transport mechanism. Two different functions of protons for the ion pump must be distinguished. On one hand, there are electrogenically bound "functional" protons, which are not transported but prerequisite for the performance of the ATP-driven half-cycle. On the other hand, protons bind to the transport sites, acting as weak congeners of K(+). There possibly are noncompetitively bound protons, affecting the enzyme activity and/or coupling between KdpA and KdpB subunits. Finally, the recently proposed Post-Albers model for the KdpFABC complex was supplemented with stoichiometry factors of 2 for K(+) and 3 for H(+), and additional inhibitory side reactions controlled by H(+) were introduced, which are relevant at pH <6.5 and/or in the absence of K(+).

Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of Acidic Fibroblast Growth Factor

PubMed Central

Jayanthi, Srinivas; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy; Furr, Mercede; Daily, Anna; Thurman, Ryan; Rutherford, Lindsay; Chandrashekar, Reena; Adams, Paul; Prudovsky, Igor; Suresh Kumar, Thallapuranam Krishnaswamy

2014-01-01

Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER) -Golgi secretory pathway. FGF1 is released through a Cu2+ - mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu2+- mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that binding of Cu2+ to the C2B domain is important for the release of FGF1 in to the extracellular medium. In this study, using a variety of biophysical studies, Cu2+ and lipid interactions of the C2B domain of Syt1were characterized. Isothermal titration calorimetry (ITC) experiments reveal that C2B domain binds to Cu2+ in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb3+ show that there are two Cu2+- binding pockets on the C2B domain, and one of these is also a Ca2+- binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu2+. The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1. PMID:25224745

Memory binding and white matter integrity in familial Alzheimer’s disease

PubMed Central

Saarimäki, Heini; Bastin, Mark E.; Londoño, Ana C.; Pettit, Lewis; Lopera, Francisco; Della Sala, Sergio; Abrahams, Sharon

2015-01-01

Binding information in short-term and long-term memory are functions sensitive to Alzheimer’s disease. They have been found to be affected in patients who meet criteria for familial Alzheimer’s disease due to the mutation E280A of the PSEN1 gene. However, only short-term memory binding has been found to be affected in asymptomatic carriers of this mutation. The neural correlates of this dissociation are poorly understood. The present study used diffusion tensor magnetic resonance imaging to investigate whether the integrity of white matter structures could offer an account. A sample of 19 patients with familial Alzheimer’s disease, 18 asymptomatic carriers and 21 non-carrier controls underwent diffusion tensor magnetic resonance imaging, neuropsychological and memory binding assessment. The short-term memory binding task required participants to detect changes across two consecutive screens displaying arrays of shapes, colours, or shape-colour bindings. The long-term memory binding task was a Paired Associates Learning Test. Performance on these tasks were entered into regression models. Relative to controls, patients with familial Alzheimer’s disease performed poorly on both memory binding tasks. Asymptomatic carriers differed from controls only in the short-term memory binding task. White matter integrity explained poor memory binding performance only in patients with familial Alzheimer’s disease. White matter water diffusion metrics from the frontal lobe accounted for poor performance on both memory binding tasks. Dissociations were found in the genu of corpus callosum which accounted for short-term memory binding impairments and in the hippocampal part of cingulum bundle which accounted for long-term memory binding deficits. The results indicate that white matter structures in the frontal and temporal lobes are vulnerable to the early stages of familial Alzheimer’s disease and their damage is associated with impairments in two memory binding functions known to be markers for Alzheimer’s disease. PMID:25762465

Memory binding and white matter integrity in familial Alzheimer's disease.

PubMed

Parra, Mario A; Saarimäki, Heini; Bastin, Mark E; Londoño, Ana C; Pettit, Lewis; Lopera, Francisco; Della Sala, Sergio; Abrahams, Sharon

2015-05-01

Binding information in short-term and long-term memory are functions sensitive to Alzheimer's disease. They have been found to be affected in patients who meet criteria for familial Alzheimer's disease due to the mutation E280A of the PSEN1 gene. However, only short-term memory binding has been found to be affected in asymptomatic carriers of this mutation. The neural correlates of this dissociation are poorly understood. The present study used diffusion tensor magnetic resonance imaging to investigate whether the integrity of white matter structures could offer an account. A sample of 19 patients with familial Alzheimer's disease, 18 asymptomatic carriers and 21 non-carrier controls underwent diffusion tensor magnetic resonance imaging, neuropsychological and memory binding assessment. The short-term memory binding task required participants to detect changes across two consecutive screens displaying arrays of shapes, colours, or shape-colour bindings. The long-term memory binding task was a Paired Associates Learning Test. Performance on these tasks were entered into regression models. Relative to controls, patients with familial Alzheimer's disease performed poorly on both memory binding tasks. Asymptomatic carriers differed from controls only in the short-term memory binding task. White matter integrity explained poor memory binding performance only in patients with familial Alzheimer's disease. White matter water diffusion metrics from the frontal lobe accounted for poor performance on both memory binding tasks. Dissociations were found in the genu of corpus callosum which accounted for short-term memory binding impairments and in the hippocampal part of cingulum bundle which accounted for long-term memory binding deficits. The results indicate that white matter structures in the frontal and temporal lobes are vulnerable to the early stages of familial Alzheimer's disease and their damage is associated with impairments in two memory binding functions known to be markers for Alzheimer's disease. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Molecular dynamics and Monte Carlo simulations for protein-ligand binding and inhibitor design.

PubMed

Cole, Daniel J; Tirado-Rives, Julian; Jorgensen, William L

2015-05-01

Non-nucleoside inhibitors of HIV reverse transcriptase are an important component of treatment against HIV infection. Novel inhibitors are sought that increase potency against variants that contain the Tyr181Cys mutation. Molecular dynamics based free energy perturbation simulations have been run to study factors that contribute to protein-ligand binding, and the results are compared with those from previous Monte Carlo based simulations and activity data. Predictions of protein-ligand binding modes are very consistent for the two simulation methods; the accord is attributed to the use of an enhanced sampling protocol. The Tyr181Cys binding pocket supports large, hydrophobic substituents, which is in good agreement with experiment. Although some discrepancies exist between the results of the two simulation methods and experiment, free energy perturbation simulations can be used to rapidly test small molecules for gains in binding affinity. Free energy perturbation methods show promise in providing fast, reliable and accurate data that can be used to complement experiment in lead optimization projects. This article is part of a Special Issue entitled "Recent developments of molecular dynamics". Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of potassium and sodium binding in vivo and in agarose samples using TQTPPI pulse sequence

NASA Astrophysics Data System (ADS)

Schepkin, Victor D.; Neubauer, Andreas; Nagel, Armin M.; Budinger, Thomas F.

2017-04-01

Potassium and sodium specific binding in vivo were explored at 21.1 T by triple quantum (TQ) magnetic resonance (MR) signals without filtration to achieve high sensitivities and precise quantifications. The pulse sequence used time proportional phase increments (TPPI). During simultaneous phase-time increments, it provided total single quantum (SQ) and TQ MR signals in the second dimension at single and triple quantum frequencies, respectively. The detection of both TQ and SQ signals was performed at identical experimental conditions and the resulting TQ signal equals 60 ± 3% of the SQ signal when all ions experience sufficient time for binding. In a rat head in vivo the TQ percentage relative to SQ for potassium is 41.5 ± 3% and for sodium is 16.1 ± 1%. These percentages were compared to the matching values in an agarose tissue model with MR relaxation times similar to those of mammalian brain tissue. The sodium TQ signal in agarose samples decreased in the presence of potassium, suggesting a competitive binding of potassium relative to sodium ions for the same binding sites. The TQTPPI signals correspond to almost two times more effective binding of potassium than sodium. In vivo, up to ∼69% of total potassium and ∼27% of total sodium can be regarded as bound or experiencing an association time in the range of several milliseconds. Experimental data analyses show that more than half of the in vivo total sodium TQ signal could be from extracellular space, which is an important factor for quantification of intracellular MR signals.

Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

PubMed

Poda, Suresh B; Kobayashi, Masakazu; Nachane, Ruta; Menon, Veena; Gandhi, Adarsh S; Budac, David P; Li, Guiying; Campbell, Brian M; Tagmose, Lena

2015-10-01

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-β-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

Investigating the interaction of anticancer drug temsirolimus with human transferrin: Molecular docking and spectroscopic approach.

PubMed

Shamsi, Anas; Ahmed, Azaj; Khan, Mohd Shahnawaz; Husain, Fohad Mabood; Amani, Samreen; Bano, Bilqees

2018-05-16

In our present study, binding between an important anti renal cancer drug temsirolimus and human transferrin (hTF) was investigated employing spectroscopic and molecular docking approach. In the presence of temsirolimus, hyper chromaticity is observed in hTF in UV spectroscopy suggestive of complex formation between hTF and temsirolimus. Fluorescence spectroscopy revealed the occurrence of quenching in hTF in the presence of temsirolimus implying complex formation taking place between hTF and temsirolimus. Further, the mode of interaction between hTF and temsirolimus was revealed to be static by fluorescence quenching analysis at 3 different temperatures. Binding constant values obtained employing fluorescence spectroscopy depicts strong interaction between hTF and temsirolimus; temsirolimus binds to hTF at 298 K with a binding constant of .32 × 10 4  M -1 implying the strength of this interaction. The negative Gibbs free energy obtained through quenching experiments is evident of the fact that the binding is spontaneous. CD spectra of hTF also showed a downward shift in the presence of temsirolimus as compared with free hTF implying complex formation between hTF and temsirolimus. Molecular docking was performed with a view to find out which residues are key players in this interaction. The importance of our study stems from the fact it will provide an insight into binding pattern of commonly administered renal cancer drug with an important protein that plays a pivotal role in many physiological processes. Copyright © 2018 John Wiley & Sons, Ltd.

Sorption of lead onto two gram-negative marine bacteria in seawater

USGS Publications Warehouse

Harvey, Ronald W.; Leckie, James O.

1985-01-01

Laboratory adsorption experiments performed at environmentally significant lead (Pb) and cell concentrations indicate that the marine bacteria examined have significant binding capacities for Pb. However, the behavior governing Pb sorption onto gram-negative bacteria in seawater may be quite complex. The sorption kinetics appear to involve two distinct phases, i.e., a rapid removal of Pb from solution within the first few minutes, followed by a slow but nearly constant removal over many hours. Also, the average binding coefficient, calculated for Pb sorption onto bacteria and a measure of binding intensity, increases with decreasing sorption density (amounts of bacteria-associated Pb per unit bacterial surface) at low cell concentrations (105 cells ml−1), but decreases with decreasing sorption density at higher cell concentrations (107 cells ml−1). The latter effect is apparently due to the production of significant amounts of extra-cellular organics at high cell concentrations that compete directly with bacterial surfaces for available lead. Lead toxicity and active uptake by marine bacteria did not appear significant at the Pb concentrations used.

Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches.

PubMed

El Bakkali-Tahéri, Nadia; Tachon, Sybille; Orio, Maylis; Bertaina, Sylvain; Martinho, Marlène; Robert, Viviane; Réglier, Marius; Tron, Thierry; Dorlet, Pierre; Simaan, A Jalila

2017-06-01

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a non heme iron(II) containing enzyme that catalyzes the final step of the ethylene biosynthesis in plants. The iron(II) ion is bound in a facial triad composed of two histidines and one aspartate (H177, D179 and H234). Several active site variants were generated to provide alternate binding motifs and the enzymes were reconstituted with copper(II). Continuous wave (cw) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopies as well as Density Functional Theory (DFT) calculations were performed and models for the copper(II) binding sites were deduced. In all investigated enzymes, the copper ion is equatorially coordinated by the two histidine residues (H177 and H234) and probably two water molecules. The copper-containing enzymes are inactive, even when hydrogen peroxide is used in peroxide shunt approach. EPR experiments and DFT calculations were undertaken to investigate substrate's (ACC) binding on the copper ion and the results were used to rationalize the lack of copper-mediated activity. Copyright © 2017 Elsevier Inc. All rights reserved.

NMR and MD Investigations of Human Galectin-1/Oligosaccharide Complexes

PubMed Central

Meynier, Christophe; Feracci, Mikael; Espeli, Marion; Chaspoul, Florence; Gallice, Philippe; Schiff, Claudine; Guerlesquin, Françoise; Roche, Philippe

2009-01-01

Abstract The specific recognition of carbohydrates by lectins plays a major role in many cellular processes. Galectin-1 belongs to a family of 15 structurally related β-galactoside binding proteins that are able to control a variety of cellular events, including cell cycle regulation, adhesion, proliferation, and apoptosis. The three-dimensional structure of galectin-1 has been solved by x-ray crystallography in the free form and in complex with various carbohydrate ligands. In this work, we used a combination of two-dimensional NMR titration experiments and molecular-dynamics simulations with explicit solvent to study the mode of interaction between human galectin-1 and five galactose-containing ligands. Isothermal titration calorimetry measurements were performed to determine their affinities for galectin-1. The contribution of the different hexopyranose units in the protein-carbohydrate interaction was given particular consideration. Although the galactose moiety of each oligosaccharide is necessary for binding, it is not sufficient by itself. The nature of both the reducing sugar in the disaccharide and the interglycosidic linkage play essential roles in the binding to human galectin-1. PMID:20006954

The cellulose binding region in Trichoderma reesei cellobiohydrolase I has a higher capacity in improving crystalline cellulose degradation than that of Penicillium oxalicum.

PubMed

Du, Jian; Zhang, Xiu; Li, Xuezhi; Zhao, Jian; Liu, Guodong; Gao, Baoyu; Qu, Yinbo

2018-06-19

Commercial cellulase preparations for lignocellulose bioconversion are mainly produced by the fungus Trichoderma reesei. The maximum cellulose conversion of T. reesei cellulase mixture was 15%-20% higher than that of Penicillium oxalicum in the hydrolysis of corncob residue and Avicel. Nevertheless, both preparations hydrolyzed more than 92% of cellulose in NaOH-mercerized Avicel. When added to Avicel hydrolysis residue that was less reactive to P. oxalicum cellulases, cellobiohydrolase I (CBH I) from T. reesei resulted in a higher cellulose conversion than its homologous proteins from P. oxalicum and Aspergillus niger at the same protein loadings. Further domain exchange experiment attributed the high hydrolytic efficiency of T. reesei CBH I to its inter-domain linker and cellulose-binding domain. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulases. Copyright © 2018 Elsevier Ltd. All rights reserved.

Carbachol dimers as homobivalent modulators of muscarinic receptors.

PubMed

Matucci, Rosanna; Nesi, Marta; Martino, Maria Vittoria; Bellucci, Cristina; Manetti, Dina; Ciuti, Elisa; Mazzolari, Angelica; Dei, Silvia; Guandalini, Luca; Teodori, Elisabetta; Vistoli, Giulio; Romanelli, Maria Novella

2016-05-15

A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing the two agonist units symmetrically connected through a methylene chain of variable length. The new compounds have been tested on the five cloned muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding studies, showing an increase in affinity by rising the number of methylene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 on CHO cells have shown that the new compounds are endowed with muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested compounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simulations, performed on the hM1 and hM2 receptors, give a sound rationalization of the experimental data revealing how these compounds are able to interact with both orthosteric and allosteric binding sites depending on the length of their connecting chain. Copyright © 2016 Elsevier Inc. All rights reserved.

Large-scale modelling of the divergent spectrin repeats in nesprins: giant modular proteins.

PubMed

Autore, Flavia; Pfuhl, Mark; Quan, Xueping; Williams, Aisling; Roberts, Roland G; Shanahan, Catherine M; Fraternali, Franca

2013-01-01

Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht-ANC-Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.

Absolute binding free energy calculations of CBClip host–guest systems in the SAMPL5 blind challenge

PubMed Central

Tofoleanu, Florentina; Pickard, Frank C.; König, Gerhard; Huang, Jing; Damjanović, Ana; Baek, Minkyung; Seok, Chaok; Brooks, Bernard R.

2016-01-01

Herein, we report the absolute binding free energy calculations of CBClip complexes in the SAMPL5 blind challenge. Initial conformations of CBClip complexes were obtained using docking and molecular dynamics simulations. Free energy calculations were performed using thermodynamic integration (TI) with soft-core potentials and Bennett’s acceptance ratio (BAR) method based on a serial insertion scheme. We compared the results obtained with TI simulations with soft-core potentials and Hamiltonian replica exchange simulations with the serial insertion method combined with the BAR method. The results show that the difference between the two methods can be mainly attributed to the van der Waals free energies, suggesting that either the simulations used for TI or the simulations used for BAR, or both are not fully converged and the two sets of simulations may have sampled difference phase space regions. The penalty scores of force field parameters of the 10 guest molecules provided by CHARMM Generalized Force Field can be an indicator of the accuracy of binding free energy calculations. Among our submissions, the combination of docking and TI performed best, which yielded the root mean square deviation of 2.94 kcal/mol and an average unsigned error of 3.41 kcal/mol for the ten guest molecules. These values were best overall among all participants. However, our submissions had little correlation with experiments. PMID:27677749

Docking, synthesis, and NMR studies of mannosyl trisaccharide ligands for DC-SIGN lectin.

PubMed

Reina, José J; Díaz, Irene; Nieto, Pedro M; Campillo, Nuria E; Páez, Juan A; Tabarani, Georges; Fieschi, Franck; Rojo, Javier

2008-08-07

DC-SIGN, a lectin, which presents at the surface of immature dendritic cells, constitutes nowadays a promising target for the design of new antiviral drugs. This lectin recognizes highly glycosylated proteins present at the surface of several pathogens such as HIV, Ebola virus, Candida albicans, Mycobacterium tuberculosis, etc. Understanding the binding mode of this lectin is a topic of tremendous interest and will permit a rational design of new and more selective ligands. Here, we present computational and experimental tools to study the interaction of di- and trisaccharides with DC-SIGN. Docking analysis of complexes involving mannosyl di- and trisaccharides and the carbohydrate recognition domain (CRD) of DC-SIGN have been performed. Trisaccharides Manalpha1,2[Manalpha1,6]Man 1 and Manalpha1,3[Manalpha1,6]Man 2 were synthesized from an orthogonally protected mannose as a common intermediate. Using these ligands and the soluble extracellular domain (ECD) of DC-SIGN, NMR experiments based on STD and transfer-NOE were performed providing additional information. Conformational analysis of the mannosyl ligands in the free and bound states was done. These studies have demonstrated that terminal mannoses at positions 2 or 3 in the trisaccharides are the most important moiety and present the strongest contact with the binding site of the lectin. Multiple binding modes could be proposed and therefore should be considered in the design of new ligands.

firestar--advances in the prediction of functionally important residues.

PubMed

Lopez, Gonzalo; Maietta, Paolo; Rodriguez, Jose Manuel; Valencia, Alfonso; Tress, Michael L

2011-07-01

firestar is a server for predicting catalytic and ligand-binding residues in protein sequences. Here, we present the important developments since the first release of firestar. Previous versions of the server required human interpretation of the results; the server is now fully automatized. firestar has been implemented as a web service and can now be run in high-throughput mode. Prediction coverage has been greatly improved with the extension of the FireDB database and the addition of alignments generated by HHsearch. Ligands in FireDB are now classified for biological relevance. Many of the changes have been motivated by the critical assessment of techniques for protein structure prediction (CASP) ligand-binding prediction experiment, which provided us with a framework to test the performance of firestar. URL: http://firedb.bioinfo.cnio.es/Php/FireStar.php.

Laboratory automation of high-quality and efficient ligand-binding assays for biotherapeutic drug development.

PubMed

Wang, Jin; Patel, Vimal; Burns, Daniel; Laycock, John; Pandya, Kinnari; Tsoi, Jennifer; DeSilva, Binodh; Ma, Mark; Lee, Jean

2013-07-01

Regulated bioanalytical laboratories that run ligand-binding assays in support of biotherapeutics development face ever-increasing demand to support more projects with increased efficiency. Laboratory automation is a tool that has the potential to improve both quality and efficiency in a bioanalytical laboratory. The success of laboratory automation requires thoughtful evaluation of program needs and fit-for-purpose strategies, followed by pragmatic implementation plans and continuous user support. In this article, we present the development of fit-for-purpose automation of total walk-away and flexible modular modes. We shared the sustaining experience of vendor collaboration and team work to educate, promote and track the use of automation. The implementation of laboratory automation improves assay performance, data quality, process efficiency and method transfer to CRO in a regulated bioanalytical laboratory environment.

Facilitated Diffusion of Transcription Factor Proteins with Anomalous Bulk Diffusion.

PubMed

Liu, Lin; Cherstvy, Andrey G; Metzler, Ralf

2017-02-16

What are the physical laws of the diffusive search of proteins for their specific binding sites on DNA in the presence of the macromolecular crowding in cells? We performed extensive computer simulations to elucidate the protein target search on DNA. The novel feature is the viscoelastic non-Brownian protein bulk diffusion recently observed experimentally. We examine the influence of the protein-DNA binding affinity and the anomalous diffusion exponent on the target search time. In all cases an optimal search time is found. The relative contribution of intermittent three-dimensional bulk diffusion and one-dimensional sliding of proteins along the DNA is quantified. Our results are discussed in the light of recent single molecule tracking experiments, aiming at a better understanding of the influence of anomalous kinetics of proteins on the facilitated diffusion mechanism.

firestar—advances in the prediction of functionally important residues

PubMed Central

Lopez, Gonzalo; Maietta, Paolo; Rodriguez, Jose Manuel; Valencia, Alfonso; Tress, Michael L.

2011-01-01

firestar is a server for predicting catalytic and ligand-binding residues in protein sequences. Here, we present the important developments since the first release of firestar. Previous versions of the server required human interpretation of the results; the server is now fully automatized. firestar has been implemented as a web service and can now be run in high-throughput mode. Prediction coverage has been greatly improved with the extension of the FireDB database and the addition of alignments generated by HHsearch. Ligands in FireDB are now classified for biological relevance. Many of the changes have been motivated by the critical assessment of techniques for protein structure prediction (CASP) ligand-binding prediction experiment, which provided us with a framework to test the performance of firestar. URL: http://firedb.bioinfo.cnio.es/Php/FireStar.php. PMID:21672959

Study of interactions of an anticancer drug neratinib with bovine serum albumin: Spectroscopic and molecular docking approach

NASA Astrophysics Data System (ADS)

Wani, Tanveer A.; Bakheit, Ahmed H.; Abounassif, M. A.; Zargar, Seema

2018-03-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.

Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach.

PubMed

Wani, Tanveer A; Bakheit, Ahmed H; Abounassif, M A; Zargar, Seema

2018-01-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.

Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach

PubMed Central

Wani, Tanveer A.; Bakheit, Ahmed H.; Abounassif, M. A.; Zargar, Seema

2018-01-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes. PMID:29564326

Mathematical modeling of vesicle drug delivery systems 2: targeted vesicle interactions with cells, tumors, and the body.

PubMed

Ying, Chong T; Wang, Juntian; Lamm, Robert J; Kamei, Daniel T

2013-02-01

Vesicles have been studied for several years in their ability to deliver drugs. Mathematical models have much potential in reducing time and resources required to engineer optimal vesicles, and this review article summarizes these models that aid in understanding the ability of targeted vesicles to bind and internalize into cancer cells, diffuse into tumors, and distribute in the body. With regard to binding and internalization, radiolabeling and surface plasmon resonance experiments can be performed to determine optimal vesicle size and the number and type of ligands conjugated. Binding and internalization properties are also inputs into a mathematical model of vesicle diffusion into tumor spheroids, which highlights the importance of the vesicle diffusion coefficient and the binding affinity of the targeting ligand. Biodistribution of vesicles in the body, along with their half-life, can be predicted with compartmental models for pharmacokinetics that include the effect of targeting ligands, and these predictions can be used in conjunction with in vivo models to aid in the design of drug carriers. Mathematical models can prove to be very useful in drug carrier design, and our hope is that this review will encourage more investigators to combine modeling with quantitative experimentation in the field of vesicle-based drug delivery.

SH2 Binding Site Protection Assay: A Method for Identification of SH2 Domain Interaction Partners by Exploiting SH2 Mediated Phosphosite Protection.

PubMed

Jadwin, Joshua A

2017-01-01

Over the last two decades there has been a significant effort in the field to characterize the phosphosite binding specificities of SH2 domains with the goal of deciphering the pY signaling code. Although high throughput studies in various formats using most SH2 domains have collectively provided a rich resource of in vitro SH2-pTyr site specificity maps, this data can only be used approximate what is happening in the cell where protein concentrations and localization are not homogenous, as they are for in vitro experiments. Here we describe an in vivo approach, SH2 site protection assay, which can capture the pTyr binding specificity of SH2 domains in the cell. The basis of this approach is SH2-pY site protection, the ability of SH2 domains to prevent the PTP-dependent dephosphorylation of their pY site binding partners. We overexpress a tracer SH2 domain in cells and quantify the change in abundance of tyrosine phosphorylated sites using MS. Since the method is performed in vivo, it has the advantage of identifying SH2-pY interactions as they occur within in the cell.

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

PubMed

Doyle, Michael L; Tian, Shin-Shay; Miller, Stephen G; Kessler, Linda; Baker, Audrey E; Brigham-Burke, Michael R; Dillon, Susan B; Duffy, Kevin J; Keenan, Richard M; Lehr, Ruth; Rosen, Jon; Schneeweis, Lumelle A; Trill, John; Young, Peter R; Luengo, Juan I; Lamb, Peter

2003-03-14

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

Approaching Pharmacological Space: Events and Components.

PubMed

Vistoli, Giulio; Pedretti, Alessandro; Mazzolari, Angelica; Testa, Bernard

2018-01-01

With a view to introducing the concept of pharmacological space and its potential applications in investigating and predicting the toxic mechanisms of xenobiotics, this opening chapter describes the logical relations between conformational behavior, physicochemical properties and binding spaces, which are seen as the three key elements composing the pharmacological space. While the concept of conformational space is routinely used to encode molecular flexibility, the concepts of property spaces and, particularly, of binding spaces are more innovative. Indeed, their descriptors can find fruitful applications (a) in describing the dynamic adaptability a given ligand experiences when inserted into a specific environment, and (b) in parameterizing the flexibility a ligand retains when bound to a biological target. Overall, these descriptors can conveniently account for the often disregarded entropic factors and as such they prove successful when inserted in ligand- or structure-based predictive models. Notably, and although binding space parameters can clearly be derived from MD simulations, the chapter will illustrate how docking calculations, despite their static nature, are able to evaluate ligand's flexibility by analyzing several poses for each ligand. Such an approach, which represents the founding core of the binding space concept, can find various applications in which the related descriptors show an impressive enhancing effect on the statistical performances of the resulting predictive models.

A Comparison of Two Single-Stranded DNA Binding Models by Mutational Analysis of APOBEC3G

PubMed Central

Shindo, Keisuke; Li, Ming; Gross, Phillip J.; Brown, William L.; Harjes, Elena; Lu, Yongjian; Matsuo, Hiroshi; Harris, Reuben S.

2012-01-01

APOBEC3G is the best known of several DNA cytosine deaminases that function to inhibit the replication of parasitic genetic elements including the lentivirus HIV. Several high-resolution structures of the APOBEC3G catalytic domain have been generated, but none reveal how this enzyme binds to substrate single-stranded DNA. Here, we constructed a panel of APOBEC3G amino acid substitution mutants and performed a series of biochemical, genetic, and structural assays to distinguish between “Brim” and “Kink” models for single-strand DNA binding. Each model predicts distinct sets of interactions between surface arginines and negatively charged phosphates in the DNA backbone. Concordant with both models, changing the conserved arginine at position 313 to glutamate abolished both catalytic and restriction activities. In support of the Brim model, arginine to glutamate substitutions at positions 213, 215, and 320 also compromised these APOBEC3G activities. Arginine to glutamate substitutions at Kink model residues 374 and 376 had smaller effects. These observations were supported by A3G catalytic domain-ssDNA chemical shift perturbation experiments. The overall data set is most consistent with the Brim model for single-stranded DNA binding by APOBEC3G. PMID:24832226

Identification of a Cholesterol-Binding Pocket in Inward Rectifier K+ (Kir) Channels

PubMed Central

Fürst, Oliver; Nichols, Colin G.; Lamoureux, Guillaume; D’Avanzo, Nazzareno

2014-01-01

Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K+ (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket. PMID:25517146

Zinc-mediated Allosteric Inhibition of Caspase-6*

PubMed Central

Velázquez-Delgado, Elih M.; Hardy, Jeanne A.

2012-01-01

Zinc and caspase-6 have independently been implicated in several neurodegenerative disorders. Depletion of zinc intracellularly leads to apoptosis by an unknown mechanism. Zinc inhibits cysteine proteases, including the apoptotic caspases, leading to the hypothesis that zinc-mediated inhibition of caspase-6 might contribute to its regulation in a neurodegenerative context. Using inductively coupled plasma optical emission spectroscopy, we observed that caspase-6 binds one zinc per monomer, under the same conditions where the zinc leads to complete loss of enzymatic activity. To understand the molecular details of zinc binding and inhibition, we performed an anomalous diffraction experiment above the zinc edge. The anomalous difference maps showed strong 5σ peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site. Zinc was not observed bound to the active site. The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family. Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation. PMID:22891250

On the role of humic acids' carboxyl groups in the binding of charged organic compounds.

PubMed

Smilek, Jiří; Sedláček, Petr; Kalina, Michal; Klučáková, Martina

2015-11-01

Interactions of humic acids (HAs) with two cationic dyes (methylene blue and rhodamine 6G) were studied using a unique combination of diffusion and partitioning studies in HAs, containing hydrogels and batch sorption experiments. In order to investigate the involvement of carboxyl groups of HAs in these interactions, all experiments were performed for both, the original lignite HAs and HAs with selectively methylated carboxyls. The results of the diffusion experiments confirm that the interactions between the solute and humic substances have a strong impact on the rate of diffusion process. Surprisingly, the effect is almost equally approved for original and methylated HAs. On the other hand, the results of batch sorption experiments show strong improvement of the sorption capacity (methylated HAs), which is explained by changed morphology of alkylated HAs. The comparison of the results of diffusion and adsorption experiments shows that the diffusion experiments simulate the transport of solutes in natural humics containing environment more reasonably. Copyright © 2015 Elsevier Ltd. All rights reserved.

Circular Dichroism of G-Quadruplex: A Laboratory Experiment for the Study of Topology and Ligand Binding

ERIC Educational Resources Information Center

Carvalho, Josue´; Queiroz, João A.; Cruz, Carla

2017-01-01

Circular dichroism (CD) has emerged as one of the standard biophysical techniques for the study of guaninequadruplex (G4) folding, cation effect, and ligand binding. The utility of this technique is based on its robustness, ease of use, and requirement of only small quantities of nucleic acid. This experiment is also extendable to the classroom…

Nuclear medium effects in structure functions of nucleon at moderate Q2

NASA Astrophysics Data System (ADS)

Haider, H.; Zaidi, F.; Sajjad Athar, M.; Singh, S. K.; Ruiz Simo, I.

2015-11-01

Recent experiments performed on inclusive electron scattering from nuclear targets have measured the nucleon electromagnetic structure functions F1 (x ,Q2), F2 (x ,Q2) and FL (x ,Q2) in 12C, 27Al, 56Fe and 64Cu nuclei. The measurements have been done in the energy region of 1 GeV2

Artificial intelligence systems based on texture descriptors for vaccine development.

PubMed

Nanni, Loris; Brahnam, Sheryl; Lumini, Alessandra

2011-02-01

The aim of this work is to analyze and compare several feature extraction methods for peptide classification that are based on the calculation of texture descriptors starting from a matrix representation of the peptide. This texture-based representation of the peptide is then used to train a support vector machine classifier. In our experiments, the best results are obtained using local binary patterns variants and the discrete cosine transform with selected coefficients. These results are better than those previously reported that employed texture descriptors for peptide representation. In addition, we perform experiments that combine standard approaches based on amino acid sequence. The experimental section reports several tests performed on a vaccine dataset for the prediction of peptides that bind human leukocyte antigens and on a human immunodeficiency virus (HIV-1). Experimental results confirm the usefulness of our novel descriptors. The matlab implementation of our approaches is available at http://bias.csr.unibo.it/nanni/TexturePeptide.zip.

BHC80 ss Critical in Suppression of Snail-LSD1 Interaction and Breast Cancer Metastasis

DTIC Science & Technology

2012-01-01

as well as Snail/LSD1 binding to the target gene promoter. The identification and functional characterization of PAPR1 will potentially help us...demonstrated that LSD1 enhances the protein stability of Snail and cooperates with Snail to induce EMT, the function of BHC80, as well as potential...identified a new protein component of the Snail/LSD1 complex, and have performed initial experiments to characterize the function of this protein, which

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

PubMed

Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

2017-09-01

Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

In vitro DNA binding studies of Aspartame, an artificial sweetener.

PubMed

Kashanian, Soheila; Khodaei, Mohammad Mehdi; Kheirdoosh, Fahimeh

2013-03-05

A number of small molecules bind directly and selectively to DNA, by inhibiting replication, transcription or topoisomerase activity. In this work the interaction of native calf thymus DNA (CT-DNA) with Aspartame (APM), an artificial sweeteners was studied at physiological pH. DNA binding study of APM is useful to understand APM-DNA interaction mechanism and to provide guidance for the application and design of new and safer artificial sweeteners. The interaction was investigated using spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD). Hypochromism and red shift are shown in UV absorption band of APM. A strong fluorescence quenching reaction of DNA to APM was observed and the binding constants (Kf) of DNA with APM and corresponding number of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be +181kJmol(-1) and +681Jmol(-1)K(-1) according to Van't Hoff equation, which indicated that reaction is predominantly entropically driven. Moreover, spectrofluorometric competition experiment and circular dichroism (CD) results are indicative of non-intercalative DNA binding nature of APM. We suggest that APM interacts with calf thymus DNA via groove binding mode with an intrinsic binding constant of 5×10(+4)M(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

Bead-based microfluidic immunoassay for diagnosis of Johne's disease

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

Wadhwa, Ashutosh; Foote, Robert; Shaw, Robert W

2012-01-01

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne s disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types ofmore » SAB, and types of magnetic beads) were optimized for a great degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested by commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads by laser-induced fluorescence. Antigen-coated magnetic beads treated with bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples obtained with the system were compared to those obtained with the flow cytometer, a high level of correlation (linear regression, r2 = 0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD. Key Words: Mycobacterium avium ssp. paratuberculosis, Johne s disease, microfluidics, lab-on-a-chip.« less

Study on performance of magnetic fluorescent nanoparticles as gene carrier and location in pig kidney cells

NASA Astrophysics Data System (ADS)

Wang, Yan; Cui, Haixin; Sun, Changjiao; Du, Wei; Cui, Jinhui; Zhao, Xiang

2013-03-01

We evaluated the performance of green fluorescent magnetic Fe3O4 nanoparticles (NPs) as gene carrier and location in pig kidney cells. When the mass ratio of NPs to green fluorescent protein plasmid DNA reached 1:16 or above, DNA molecules can be combined completely with NPs, which indicates that the NPs have good ability to bind negative DNA. Atomic force microscopy (AFM) experiments were carried out to investigate the binding mechanism between NPs and DNA. AFM images show that individual DNA strands come off of larger pieces of netlike agglomerations and several spherical nanoparticles are attached to each individual DNA strand and interact with each other. The pig kidney cells were labelled with membrane-specific red fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and nucleus-specific blue fluorescent dye 4',6-diamidino-2-phenylindole dihydrochloride. We found that green fluorescent nanoparticles can past the cell membrane and spread throughout the interior of the cell. The NPs seem to locate more frequently in the cytoplasm than in the nucleus.

Dry immunochemical sensor for the detection of PETN vapor

NASA Astrophysics Data System (ADS)

Lukens, Herbert Richard

1992-05-01

Giaever (1973) showed that an indium semi-mirror coated with a monolayer of a substance would undergo reduced optical reflectance after incubation with a solution of the substance's antibody. He was able to see the effect with the naked eye. Lukens and Williams (1977) reversed the process by first attaching the target substance's antibody to the semi-mirror, after which the device would show a decline in optical density when exposed to a solution of the target substance. Lukens and Williams (1982) subsequently found that the device could also be used as an immunochemical film badge (IFB) to detect an airborne target substance. Early efforts to develop the IFB for the detection of airborne substances were plagued by such a high degree of performance variability that there were doubts in some quarters that an airborne target substance could bind to its antibody until Lukens (1990) demonstrated such binding in experiments with radiolabeled cocaine and morphine. Recently improved semi-mirrors and densitometry have been obtained and have led to improved performance of IFB's. As shown in this paper, IFB's can now be constructed that detect PETN vapor in a few seconds.

Paradoxical roles of hydrogen in electrochemical performance of graphene: High rate capacity and atomistic origins

DOE PAGES

Ye, Jianchao C.; Ong, Mitchell T.; Heo, Tae Wook; ...

2015-11-05

Atomic hydrogen exists ubiquitously in graphene materials made by chemical methods. Yet determining the effect of hydrogen on the electrochemical performance of graphene remains a significant challenge. Here we report the experimental observations of high rate capacity in hydrogen-treated 3-dimensional (3D) graphene nanofoam electrodes for lithium ion batteries. Structural and electronic characterization suggests that defect sites and hydrogen play synergistic roles in disrupting sp 2 graphene to facilitate fast lithium transport and reversible surface binding, as evidenced by the fast charge-transfer kinetics and increased capacitive contribution in hydrogen-treated 3D graphene. In concert with experiments, multiscale calculations reveal that defect complexesmore » in graphene are prerequisite for low-temperature hydrogenation, and that the hydrogenation of defective or functionalized sites at strained domain boundaries plays a beneficial role in improving rate capacity by opening gaps to facilitate easier Li penetration. Additional reversible capacity is provided by enhanced lithium binding near hydrogen-terminated edge sites. Furthermore, these findings provide qualitative insights in helping the design of graphene-based materials for high-power electrodes.« less

Universal roles of hydrogen in electrochemical performance of graphene: high rate capacity and atomistic origins

PubMed Central

Ye, Jianchao; Ong, Mitchell T.; Heo, Tae Wook; Campbell, Patrick G.; Worsley, Marcus A.; Liu, Yuanyue; Shin, Swanee J.; Charnvanichborikarn, Supakit; Matthews, Manyalibo J.; Bagge-Hansen, Michael; Lee, Jonathan R.I.; Wood, Brandon C.; Wang, Y. Morris

2015-01-01

Atomic hydrogen exists ubiquitously in graphene materials made by chemical methods. Yet determining the effect of hydrogen on the electrochemical performance of graphene remains a significant challenge. Here we report the experimental observations of high rate capacity in hydrogen-treated 3-dimensional (3D) graphene nanofoam electrodes for lithium ion batteries. Structural and electronic characterization suggests that defect sites and hydrogen play synergistic roles in disrupting sp2 graphene to facilitate fast lithium transport and reversible surface binding, as evidenced by the fast charge-transfer kinetics and increased capacitive contribution in hydrogen-treated 3D graphene. In concert with experiments, multiscale calculations reveal that defect complexes in graphene are prerequisite for low-temperature hydrogenation, and that the hydrogenation of defective or functionalized sites at strained domain boundaries plays a beneficial role in improving rate capacity by opening gaps to facilitate easier Li penetration. Additional reversible capacity is provided by enhanced lithium binding near hydrogen-terminated edge sites. These findings provide qualitative insights in helping the design of graphene-based materials for high-power electrodes. PMID:26536830

The influence of aging on attentional refreshing and articulatory rehearsal during working memory on later episodic memory performance.

PubMed

Loaiza, Vanessa M; McCabe, David P

2013-01-01

We investigated age-related changes in two proposed mechanisms of maintenance in working memory, articulatory rehearsal, and attentional refreshing, by examining the consequences of manipulating the opportunity for each on delayed recall. Both experiments utilized modified operation span tasks to vary the opportunity for articulatory rehearsal (Experiment 1) and attentional refreshing opportunities (Experiment 2). In both experiments, episodic memory was tested for items that had been initially studied during the respective operation span task. Older adults' episodic memory benefited less from opportunities for refreshing than younger adults. In contrast, articulatory rehearsal opportunities did not influence episodic memory for either age group. The results suggest that attentional refreshing, and not articulatory rehearsal, is important during working memory in order to bind more accessible traces at later tests, which appears to be more deficient in older adults than younger adults.

Microplate-Based Method for High-Throughput Screening (HTS) of Chromatographic Conditions Studies for Recombinant Protein Purification.

PubMed

Carvalho, Rimenys J; Cruz, Thayana A

2018-01-01

High-throughput screening (HTS) systems have emerged as important tools to provide fast and low cost evaluation of several conditions at once since it requires small quantities of material and sample volumes. These characteristics are extremely valuable for experiments with large number of variables enabling the application of design of experiments (DoE) strategies or simple experimental planning approaches. Once, the capacity of HTS systems to mimic chromatographic purification steps was established, several studies were performed successfully including scale down purification. Here, we propose a method for studying different purification conditions that can be used for any recombinant protein, including complex and glycosylated proteins, using low binding filter microplates.

HOCOMOCO: towards a complete collection of transcription factor binding models for human and mouse via large-scale ChIP-Seq analysis.

PubMed

Kulakovskiy, Ivan V; Vorontsov, Ilya E; Yevshin, Ivan S; Sharipov, Ruslan N; Fedorova, Alla D; Rumynskiy, Eugene I; Medvedeva, Yulia A; Magana-Mora, Arturo; Bajic, Vladimir B; Papatsenko, Dmitry A; Kolpakov, Fedor A; Makeev, Vsevolod J

2018-01-04

We present a major update of the HOCOMOCO collection that consists of patterns describing DNA binding specificities for human and mouse transcription factors. In this release, we profited from a nearly doubled volume of published in vivo experiments on transcription factor (TF) binding to expand the repertoire of binding models, replace low-quality models previously based on in vitro data only and cover more than a hundred TFs with previously unknown binding specificities. This was achieved by systematic motif discovery from more than five thousand ChIP-Seq experiments uniformly processed within the BioUML framework with several ChIP-Seq peak calling tools and aggregated in the GTRD database. HOCOMOCO v11 contains binding models for 453 mouse and 680 human transcription factors and includes 1302 mononucleotide and 576 dinucleotide position weight matrices, which describe primary binding preferences of each transcription factor and reliable alternative binding specificities. An interactive interface and bulk downloads are available on the web: http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco11. In this release, we complement HOCOMOCO by MoLoTool (Motif Location Toolbox, http://molotool.autosome.ru) that applies HOCOMOCO models for visualization of binding sites in short DNA sequences. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.

PubMed

Gater, Deborah L; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

2014-11-18

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Photochemical and DFT studies on DNA-binding ability and antibacterial activity of lanthanum(III)-phenanthroline complex

NASA Astrophysics Data System (ADS)

Niroomand, Sona; Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Jahani, Shohreh; Moodi, Asieh

2017-02-01

The binding of the lanthanum(III) complex containing 1,10-phenanthroline (phen), [La(phen)3Cl3·OH2], to DNA is investigated by absorption and emission methods. This complex shows absorption decreasing in a charge transfer band, and fluorescence decrement when it binds to DNA. Electronic absorption spectroscopy (UV-Vis), fluorescence spectra, iodide quenching experiments, salt effect and viscosity measurements, ethidium bromide (EB) competition test, circular dichroism (CD) spectra as well as variable temperature experiments indicate that the La(III) complex binds to fish salmon (FS) DNA, presumably via groove binding mode. The binding constants (Kb) of the La(III) complex with DNA is (2.55 ± 0.02) × 106 M-1. Furthermore, the binding site size, n, the Stern-Volmer constant KSV and thermodynamic parameters; enthalpy change (ΔH0) and entropy change (ΔS0) and Gibb's free energy (ΔG0), are calculated according to relevant fluorescent data and the Van't Hoff equation. The La(III) complex has been screened for its antibacterial activities by the disc diffusion method. Also, in order to supplement the experimental findings, DFT computation and NBO analysis are carried out.

Exploring the site-selective binding of jatrorrhizine to human serum albumin: spectroscopic and molecular modeling approaches.

PubMed

Mi, Ran; Hu, Yan-Jun; Fan, Xiao-Yang; Ouyang, Yu; Bai, Ai-Min

2014-01-03

This paper exploring the site-selective binding of jatrorrhizine to human serum albumin (HSA) under physiological conditions (pH=7.4). The investigation was carried out using fluorescence spectroscopy, UV-vis spectroscopy, and molecular modeling. The results of fluorescence quenching and UV-vis absorption spectra experiments indicated the formation of the complex of HSA-jatrorrhizine. Binding parameters calculating from Stern-Volmer method and Scatchard method were calculated at 298, 304 and 310 K, with the corresponding thermodynamic parameters ΔG, ΔH and ΔS as well. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that jatrorrhizine bind to HSA with the binding affinities of the order 10(4) L mol(-1). The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for jatrorrhizine-HSA association. Site marker competitive displacement experiments and molecular modeling calculation demonstrating that jatrorrhizine is mainly located within the hydrophobic pocket of the subdomain IIIA of HSA. Furthermore, the synchronous fluorescence spectra suggested that the association between jatrorrhizine and HSA changed molecular conformation of HSA. Copyright © 2013. Published by Elsevier B.V.

Binding of Phenazinium Dye Safranin T to Polyriboadenylic Acid: Spectroscopic and Thermodynamic Study

PubMed Central

Roy, Snigdha; Das, Suman

2014-01-01

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na+] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure. PMID:24498422

Binding of phenazinium dye safranin T to polyriboadenylic acid: spectroscopic and thermodynamic study.

PubMed

Pradhan, Ankur Bikash; Haque, Lucy; Roy, Snigdha; Das, Suman

2014-01-01

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na⁺] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.

Melatonin receptors: Current status, facts, and hypothesis

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

Stankov, B.; Reiter, R.J.

Great progress has been made in the identification of melatonin binding sites, commonly identified as melatonin receptors by many authors, in recent years. The bulk of these studies have investigated the sites using either autoradiographic and biochemical techniques with the majority of the experiments being done on the rat, Djungarian and Syrian hamster, and sheep, although human tissue has also been employed. Many of the studies have identified melatonin binding in the central nervous system with either tritium- or iodine-labelled ligands. The latter ligand seems to provide the most reproducible and consistent data. Of the central neural tissues examined, themore » suprachiasmatic nuclei are most frequently mentioned as a location for melatonin binding sites although binding seems to be widespread in the brain. The other tissue that has been prominently mentioned as a site for melatonin binding is the pars tuberalis of the anterior pituitary gland. There may be time-dependent variations in melatonin binding densities in both neural and pituitary gland tissue. Very few attempts have been made to identify melatonin binding outside of the central nervous system despite the widespread actions of melatonin. Preliminary experiments have been carried out on the intracellular second messengers which mediate the actions of melatonin.« less

Probing the binding of flavonoids to catalase by molecular spectroscopy

NASA Astrophysics Data System (ADS)

Zhu, Jingfeng; Zhang, Xia; Li, Daojin; Jin, Jing

2007-10-01

The binding of flavonoids (quercetin and myricetin) to catalase was investigated by fluorescence and circular dichroism (CD) techniques under physiological conditions. The binding parameters and binding mode between flavonoids and catalase were determined, and the results of synchronous fluorescence spectra and CD indicated a conformational change of catalase with addition of flavonoids. The effect of both Cu 2+ and vitamin C on the binding constant of flavonoid-catalase was also examined. The experiment data show that the difference of the structure characteristics of quercetin and myricetin has a significant effect on their binding affinity for catalase.

Evaluation of Phosphatidylserine-Binding Peptides Radiolabeled with Fluorine 18 for in vivo Imaging of Apoptosis

NASA Astrophysics Data System (ADS)

Kapty, Janice Sarah

We currently do not have a clinical method to directly assess apoptosis induced by cancer therapies. Phosphatidylserine (PS) is an attractive target for imaging apoptosis since it is on the exterior of the apoptotic cells and PS externalization is an early marker of apoptosis. PS-binding peptides are an attractive option for developing an imaging probe to detect apoptosis using positron emission tomography. In this study we evaluated binding characteristics of PS-binding peptides for ability to bind to PS, radiolabeled PS-binding peptides with fluorine-18, and performed in vitro and in vivo analysis of 18F radiolabeled PS-binding peptides including biodistribution analysis and dynamic PET imaging in a murine tumor model of apoptosis. Four peptides were evaluated for PS binding characteristics using a plate based assay system, a liposome mimic of cell membrane PS presentation, and a cell assay of apoptosis. The results indicate that all four peptides bind to PS and are specific to apoptotic cells. The widely used 18 F prosthetic group N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) and the recently developed N-[6-(4-[ 18F]fluorobenzylidene) aminooxyhexyl]maleimide ([18F]FBAM) were investigated for radiolabeling of two representative phosphatidylserine-binding peptides. The prosthetic groups were compared with respect to required reaction conditions for optimum labeling, radiolabeling yield and chemoselectivity. The N-terminus labeled product produced by reaction of [18F]SFB with binding peptide LIKKPF was produced in 18% radiochemical yield while no N-terminus labeled product could be isolated following [18F]SFB reaction with PDGLSR. When the peptides were modified by addition of a cysteine residue at the N-terminus they provided almost quantitative radiochemical yields with [18F]FBAM. Results indicate that for the peptides in this study, [18F]FBAM is a more useful prosthetic group compared to [18F]SFB due to its excellent chemo-selectivity and high radiochemical yield. We report the first experiments where PS-binding peptides were radiolabeled with 18F and evaluated as possible radiotracers for imaging apoptosis. We investigated two radio-peptides ([ 18F]FBAM-CLIKKPF and [18F]FBAM-CPGDLSR) in vitro and in vivo as possible radiotracers able to bind to apoptotic cells and to image chemotherapy induced apoptosis.

The Binding of Silibinin, the Main Constituent of Silymarin, to Site I on Human Serum Albumin.

PubMed

Yamasaki, Keishi; Sato, Hiroki; Minagoshi, Saori; Kyubun, Karin; Anraku, Makoto; Miyamura, Shigeyuki; Watanabe, Hiroshi; Taguchi, Kazuaki; Seo, Hakaru; Maruyama, Toru; Otagiri, Masaki

2017-01-01

Silibinin is the main constituent of silymarin, an extract from the seeds of milk thistle (Silybum marianum). Because silibinin has many pharmacological activities, extending its clinical use in the treatment of a wider variety of diseases would be desirable. In this study, we report on the binding of silibinin to plasma proteins, an issue that has not previously been extensively studied. The findings indicated that silibinin mainly binds to human serum albumin (HSA). Mutual displacement experiments using ligands that primarily bind to sites I and II clearly revealed that silibinin binds tightly and selectively to site I (subsites Ia and/or Ic) of HSA, which is located in subdomain IIA. Thermodynamic analyses suggested that hydrogen bonding and van der Waals interactions are major contributors to silibinin-HSA interactions. Furthermore, the binding of silibinin to HSA was found to be decreased with increasing ionic strength and detergent concentration of the media, suggesting that electrostatic and hydrophobic interactions are involved in the binding. Trp214 and Arg218 were identified as being involved in the binding of silibinin to site I, based on binding experiments using chemically modified- and mutant-HSAs. In conclusion, the available evidence indicates that silibinin binds to the region close to Trp214 and Arg218 in site I of HSA with assistance by multiple forces and can displace site I drugs (e.g., warfarin or iodipamide), but not site II drugs (e.g., ibuprofen).

Surface expression and CEA binding of hnRNP M4 protein in HT29 colon cancer cells.

PubMed

Laguinge, Luciana; Bajenova, Olga; Bowden, Emma; Sayyah, Jacqueline; Thomas, Peter; Juhl, Hartmut

2005-01-01

Carcinoembryonic antigen (CEA) has been shown to participate in the progression and metastatic growth of colorectal cancer. However, its biological function remains elusive. Recently, we found that CEA protects colon cancer cells from undergoing apoptosis, suggesting a complex role that includes signal transduction activity. Additionally, it was reported that CEA binds to Kupffer cells and macrophages to a membrane-anchored homolog of heterogeneous nuclear protein M4 (hnRNP M4), which subsequently was named CEA-receptor (CEAR). Cytoplasmatic and membranous expression of CEAR in CEA-positive colon cancer tissues prompted us to analyze the CEA-CEAR interaction in HT29 colon cancer cells. Both, CEA and CEAR were found on the cell surface of HT29 cells, as demonstrated by confocal microscopy. Imaging analysis suggested co-localization and, thus, interaction of both molecules. To confirm this observation, immunoprecipitation experiments and Western blot analysis were performed and indicated binding of CEA and CEAR. Immunoprecipitation of CEA resulted in a pull down of CEAR. The pull down of CEAR correlated with the amount of CEA as demonstrated by ribozyme targeting of CEA. Finally, external treatment of HT29 cells with soluble CEA induced tyrosine phosphorylation of CEAR, suggesting a CEA-dependent role of CEAR in signal transduction. Future experiments will elucidate whether the CEA-CEAR interaction is involved in CEA's antiapoptotic role and mediates the prometastatic properties of CEA in colon cancer cells.

Sequence-specific binding of counterions to B-DNA

PubMed Central

Denisov, Vladimir P.; Halle, Bertil

2000-01-01

Recent studies by x-ray crystallography, NMR, and molecular simulations have suggested that monovalent counterions can penetrate deeply into the minor groove of B form DNA. Such groove-bound ions potentially could play an important role in AT-tract bending and groove narrowing, thereby modulating DNA function in vivo. To address this issue, we report here 23Na magnetic relaxation dispersion measurements on oligonucleotides, including difference experiments with the groove-binding drug netropsin. The exquisite sensitivity of this method to ions in long-lived and intimate association with DNA allows us to detect sequence-specific sodium ion binding in the minor groove AT tract of three B-DNA dodecamers. The sodium ion occupancy is only a few percent, however, and therefore is not likely to contribute importantly to the ensemble of B-DNA structures. We also report results of ion competition experiments, indicating that potassium, rubidium, and cesium ions bind to the minor groove with similarly weak affinity as sodium ions, whereas ammonium ion binding is somewhat stronger. The present findings are discussed in the light of previous NMR and diffraction studies of sequence-specific counterion binding to DNA. PMID:10639130

Age-related differences in associative memory: the role of sensory decline.

PubMed

Naveh-Benjamin, Moshe; Kilb, Angela

2014-09-01

Numerous studies show age-related decline in episodic memory. One of the explanations for this decline points to older adults' deficit in associative memory, reflecting the difficulties they have in binding features of episodes into cohesive entities and retrieving these bindings. Here, we evaluate the degree to which this deficit may be mediated by sensory loss associated with increased age. In 2 experiments, young adults studied word pairs that were degraded at encoding either visually (Experiment 1) or auditorily (Experiment 2). We then tested their memory for both the component words and the associations with recognition tests. For both experiments, young adults under nondegraded conditions showed an advantage in associative over item memory, relative to a group of older adults. In contrast, under perceptually degraded conditions younger adults performed similarly to the older adults who were tested under nondegraded conditions. More specifically, under perceptual degradation, young adults' associative memory declined and their component memory improved somewhat, resulting in an associative deficit, similar to that shown by older adults. This evidence is consistent with a sensory acuity decline in old age being one mediator in the associative deficit of older adults. These results broaden our understanding of age-related memory changes and how sensory and cognitive processes interact to shape these changes. The theoretical implications of these results are discussed with respect to mechanisms underlying age-related changes in episodic memory and resource tradeoffs in the encoding of component and associative memory. PsycINFO Database Record (c) 2014 APA, all rights reserved.

Strongly Interacting Fermi Gases: Non-Equilibrium Dynamics and Dimensional Crossover

NASA Astrophysics Data System (ADS)

Sommer, Ariel

2015-05-01

Strongly interacting atomic Fermi gases near Feshbach resonances give access to a rich variety of phenomena in many-fermion physics and superfluidity. This flexible and microscopically well-characterized system provides a pristine platform in which to benchmark many-body theories. I will describe three experiments on gases of fermionic 6Li atoms. In the first experiment, we study spin transport in the return to equilibrium after a spin excitation. From the dynamics near equilibrium, we obtain spin transport coefficients over a range of temperatures and interaction strengths, and observe quantum-limited spin diffusion at unitarity. In separate experiments, we study the effect of dimensionality on the binding of pairs of fermions. We tune the system from three to two dimensions by adjusting the strength of a one-dimensional optical lattice, and measure the binding energy of fermion pairs using radio-frequency spectroscopy. In a third set of experiments, we study nonlinear excitations of a fermionic superfluid. Imprinting a phase jump on the superfluid order parameter causes a long-lived, localized density depletion that oscillates through the cloud. We measure the oscillation period and find that it corresponds to an emergent particle with an effective mass of up to several hundred times the bare mass. This excitation has been identified as a solitonic vortex that results from the decay of a planar soliton. This work was performed at the Massachusetts Institute of Technology under the supervision of Prof. Martin Zwierlein.

CELF1 preferentially binds to exon-intron boundary and regulates alternative splicing in HeLa cells.

PubMed

Xia, Heng; Chen, Dong; Wu, Qijia; Wu, Gang; Zhou, Yanhong; Zhang, Yi; Zhang, Libin

2017-09-01

The current RIP-seq approach has been developed for the identification of genome-wide interaction between RNA binding protein (RBP) and the bound RNA transcripts, but still rarely for identifying its binding sites. In this study, we performed RIP-seq experiments in HeLa cells using a monoclonal antibody against CELF1. Mapping of the RIP-seq reads showed a biased distribution at the 3'UTR and intronic regions. A total of 15,285 and 1384 CELF1-specific sense and antisense peaks were identified using the ABLIRC software tool. Our bioinformatics analyses revealed that 5' and 3' splice site motifs and GU-rich motifs were highly enriched in the CELF1-bound peaks. Furthermore, transcriptome analyses revealed that alternative splicing was globally regulated by CELF1 in HeLa cells. For example, the inclusion of exon 16 of LMO7 gene, a marker gene of breast cancer, is positively regulated by CELF1. Taken together, we have shown that RIP-seq data can be used to decipher RBP binding sites and reveal an unexpected landscape of the genome-wide CELF1-RNA interactions in HeLa cells. In addition, we found that CELF1 globally regulates the alternative splicing by binding the exon-intron boundary in HeLa cells, which will deepen our understanding of the regulatory roles of CELF1 in the pre-mRNA splicing process. Copyright © 2017 Elsevier B.V. All rights reserved.

FecB, a periplasmic ferric-citrate transporter from E. coli, can bind different forms of ferric-citrate as well as a wide variety of metal-free and metal-loaded tricarboxylic acids.

PubMed

Banerjee, Sambuddha; Paul, Subrata; Nguyen, Leonard T; Chu, Byron C H; Vogel, Hans J

2016-01-01

The Escherichia coli Fec system, consisting of an outer membrane receptor (FecA), a periplasmic substrate binding protein (FecB) and an inner membrane permease-ATPase type transporter (FecC/D), plays an important role in the uptake and transport of Fe(3+)-citrate. Although several FecB sequences from various organisms have been reported, there are no biophysical or structural data available for this protein to date. In this work, using isothermal titration calorimetry (ITC), we report for the first time the ability of FecB to bind different species of Fe(3+)-citrate as well as other citrate complexes with trivalent (Ga(3+), Al(3+), Sc(3+) and In(3+)) and a representative divalent metal ion (Mg(2+)) with low μM affinity. Interestingly, ITC experiments with various iron-free di- and tricarboxylic acids show that FecB can bind tricarboxylates with μM affinity but not biologically relevant dicarboxylates. The ability of FecB to bind with metal-free citrate is also observed in (1)H,(15)N HSQC-NMR titration experiments reported here at two different pH values. Further, differential scanning calorimetry (DSC) experiments indicate that the ligand-bound form of FecB has greater thermal stability than ligand-free FecB under all pH and ligand conditions tested, which is consistent with the idea of domain closure subsequent to ligand binding for this type of periplasmic binding proteins.

Three-Dimensional Structure and Biophysical Characterization of Staphylococcus aureus Cell Surface Antigen-Manganese Transporter MntC

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

Gribenko, Alexey; Mosyak, Lidia; Ghosh, Sharmistha

MntC is a metal-binding protein component of the Mn 2 +-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensionalmore » structure of the protein was solved by X-ray crystallography at 2.2 Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn 2 +-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn 2 +-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium–hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn 2 +.« less

Use of 2-(/sup 125/I)iodomelatonin to characterize melatonin binding sites in chicken retina

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

Dubocovich, M.L.; Takahashi, J.S.

2-(/sup 125/I)Iodomelatonin binds with high affinity to a site possessing the pharmacological characteristics of a melatonin receptor in chicken retinal membranes. The specific binding of 2-(/sup 125/I)iodomelatonin is stable, saturable, and reversible. Saturation experiments indicated that 2-(/sup 125/I)iodomelatonin labeled a single class of sites with an affinity constant (Kd) of 434 +/- 56 pM and a total number of binding sites (Bmax) of 74.0 +/- 13.6 fmol/mg of protein. The affinity constant obtained from kinetic analysis was in close agreement with that obtained in saturation experiments. Competition experiments showed a monophasic reduction of 2-(/sup 125/I)iodomelatonin binding with a pharmacological ordermore » of indole amine affinities characteristic of a melatonin receptor: 2-iodomelatonin greater than 6-chloromelatonin greater than or equal to melatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 6-hydroxymelatonin greater than or equal to 6-methoxymelatonin much greater than N-acetyltryptamine greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine greater than 5-hydroxytryptamine (inactive). The affinities of these melatonin analogs in competing for 2-(/sup 125/I)iodomelatonin binding sites were correlated closely with their potencies for inhibition of the calcium-dependent release of (3H)dopamine from chicken and rabbit retinas, indicating association of the binding site with a functional response regulated by melatonin. The results indicate that 2-(/sup 125/I)iodomelatonin is a selective, high-affinity radioligand for the identification and characterization of melatonin receptor sites.« less

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

PubMed

Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P

1996-10-01

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.

A HTML5 open source tool to conduct studies based on Libet’s clock paradigm

PubMed Central

Garaizar, Pablo; Cubillas, Carmelo P.; Matute, Helena

2016-01-01

Libet’s clock is a well-known procedure in experiments in psychology and neuroscience. Examples of its use include experiments exploring the subjective sense of agency, action-effect binding, and subjective timing of conscious decisions and perceptions. However, the technical details of the apparatus used to conduct these types of experiments are complex, and are rarely explained in sufficient detail as to guarantee an exact replication of the procedure. With this in mind, we developed Labclock Web, a web tool designed to conduct online and offline experiments using Libet’s clock. After describing its technical features, we explain how to configure specific experiments using this tool. Its degree of accuracy and precision in the presentation of stimuli has been technically validated, including the use of two cognitive experiments conducted with voluntary participants who performed the experiment both in our laboratory and via the Internet. Labclock Web is distributed without charge under a free software license (GPLv3) since one of our main objectives is to facilitate the replication of experiments and hence the advancement of knowledge in this area. PMID:27623167

A HTML5 open source tool to conduct studies based on Libet's clock paradigm.

PubMed

Garaizar, Pablo; Cubillas, Carmelo P; Matute, Helena

2016-09-13

Libet's clock is a well-known procedure in experiments in psychology and neuroscience. Examples of its use include experiments exploring the subjective sense of agency, action-effect binding, and subjective timing of conscious decisions and perceptions. However, the technical details of the apparatus used to conduct these types of experiments are complex, and are rarely explained in sufficient detail as to guarantee an exact replication of the procedure. With this in mind, we developed Labclock Web, a web tool designed to conduct online and offline experiments using Libet's clock. After describing its technical features, we explain how to configure specific experiments using this tool. Its degree of accuracy and precision in the presentation of stimuli has been technically validated, including the use of two cognitive experiments conducted with voluntary participants who performed the experiment both in our laboratory and via the Internet. Labclock Web is distributed without charge under a free software license (GPLv3) since one of our main objectives is to facilitate the replication of experiments and hence the advancement of knowledge in this area.

Quantitative structure-activity relationship and molecular docking of artemisinin derivatives to vascular endothelial growth factor receptor 1.

PubMed

Saeed, Mohamed E M; Kadioglu, Onat; Seo, Ean-Jeong; Greten, Henry Johannes; Brenk, Ruth; Efferth, Thomas

2015-04-01

The antimalarial drug artemisinin has been shown to exert anticancer activity through anti-angiogenic effects. For further drug development, it may be useful to have derivatives with improved anti-angiogenic properties. We performed molecular docking of 52 artemisinin derivatives to vascular endothelial growth factor receptors (VEGFR1, VEGFR2), and VEGFA ligand using Autodock4 and AutodockTools-1.5.7.rc1 using the Lamarckian genetic algorithm. Quantitative structure-activity relationship (QSAR) analyses of the compounds prepared by Corina Molecular Networks were performed using the Molecular Operating Environment MOE 2012.10. A statistically significant inverse relationship was obtained between in silico binding energies to VEGFR1 and anti-angiogenic activity in vivo of a test-set of artemisinin derivatives (R=-0.843; p=0.035). This served as a control experiment to validate molecular docking predicting anti-angiogenc effects. Furthermore, 52 artemisinin derivatives were docked to VEGFR1 and in selected examples also to VEGFR2 and VEGFA. Higher binding affinities were calculated for receptors than for the ligand. The best binding affinities to VEGFR1 were found for an artemisinin dimer, 10-dihydroartemisinyl-2-propylpentanoate, and dihydroartemisinin α-hemisuccinate sodium salt. QSAR analyses revealed significant relationships between VEGFR1 binding energies and defined molecular descriptors of 35 artemisinins assigned to the training set (R=0.0848, p<0.0001) and 17 derivatives assigned to the test set (R=0.761, p<0.001). Molecular docking and QSAR calculations can be used to identify novel artemisinin derivatives with anti-angiogenic effects. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Interpreting linear support vector machine models with heat map molecule coloring

PubMed Central

2011-01-01

Background Model-based virtual screening plays an important role in the early drug discovery stage. The outcomes of high-throughput screenings are a valuable source for machine learning algorithms to infer such models. Besides a strong performance, the interpretability of a machine learning model is a desired property to guide the optimization of a compound in later drug discovery stages. Linear support vector machines showed to have a convincing performance on large-scale data sets. The goal of this study is to present a heat map molecule coloring technique to interpret linear support vector machine models. Based on the weights of a linear model, the visualization approach colors each atom and bond of a compound according to its importance for activity. Results We evaluated our approach on a toxicity data set, a chromosome aberration data set, and the maximum unbiased validation data sets. The experiments show that our method sensibly visualizes structure-property and structure-activity relationships of a linear support vector machine model. The coloring of ligands in the binding pocket of several crystal structures of a maximum unbiased validation data set target indicates that our approach assists to determine the correct ligand orientation in the binding pocket. Additionally, the heat map coloring enables the identification of substructures important for the binding of an inhibitor. Conclusions In combination with heat map coloring, linear support vector machine models can help to guide the modification of a compound in later stages of drug discovery. Particularly substructures identified as important by our method might be a starting point for optimization of a lead compound. The heat map coloring should be considered as complementary to structure based modeling approaches. As such, it helps to get a better understanding of the binding mode of an inhibitor. PMID:21439031

Molecular recognition of nucleotides in micelles and the development and expansion of a chemistry outreach program

NASA Astrophysics Data System (ADS)

Schechinger, Linda Sue

I. To investigate the delivery of nucleotide-based drugs, we are studying molecular recognition of nucleotide derivatives in environments that are similar to cell membranes. The Nowick group previously discovered that membrane-like surfactant micelles tetradecyltrimethylammonium bromide (TTAB) micelle facilitate molecular of adenosine monophosphate (AMP) recognition. The micelles bind nucleotides by means of electrostatic interactions and hydrogen bonding. We observed binding by following 1H NMR chemical shift changes of unique hexylthymine protons upon addition of AMP. Cationic micelles are required for binding. In surfactant-free or sodium dodecylsulfate solutions, no hydrogen bonding is observed. These observations suggest that the cationic surfactant headgroups bind the nucleotide phosphate group, while the intramicellar base binds the nucleotide base. The micellar system was optimized to enhance binding and selectivity for adenosine nucleotides. The selectivity for adenosine and the number of phosphate groups attached to the adenosine were both investigated. Addition of cytidine, guanidine, or uridine monophosphates, results in no significant downfield shifting of the NH resonance. Selectivity for the phosphate is limited, since adenosine mono-, di-, and triphosphates all have similar binding constants. We successfully achieved molecular recognition of adenosine nucleotides in micellar environments. There is significant difference in the binding interactions between the adenosine nucleotides and three other natural nucleotides. II. The UCI Chemistry Outreach Program (UCICOP) addresses the declining interest of the nations youth for science. UCICOP brings fun and exciting chemistry experiments to local high schools, to remind students that science is fun and has many practical uses. Volunteer students and alumni of UCI perform the demonstrations using scripts and material provided by UCICOP. The preparation of scripts and materials is done by two coordinators. These coordinators organize the program and provide continuity to the program. The success of UCICOP can be measured by the high praise and gratitude expressed by the teachers, students and volunteers.

Abnormal prefrontal and parietal activity linked to deficient active binding in working memory in schizophrenia.

PubMed

Grot, Stéphanie; Légaré, Virginie Petel; Lipp, Olivier; Soulières, Isabelle; Dolcos, Florin; Luck, David

2017-10-01

Working memory deficits have been widely reported in schizophrenia, and may result from inefficient binding processes. These processes, and their neural correlates, remain understudied in schizophrenia. Thus, we designed an FMRI study aimed at investigating the neural correlates of both passive and active binding in working memory in schizophrenia. Nineteen patients with schizophrenia and 23 matched controls were recruited to perform a working memory binding task, in which they were instructed to memorize three letters and three spatial locations. In the passive binding condition, letters and spatial locations were directly presented as bound. Conversely, in the active binding condition, words and spatial locations were presented as separated, and participants were instructed to intentionally create associations between them. Patients exhibited a similar performance to the controls for the passive binding condition, but a significantly lower performance for the active binding. FMRI analyses revealed that this active binding deficit was related to aberrant activity in the posterior parietal cortex and the ventrolateral prefrontal cortex. This study provides initial evidence of a specific deficit for actively binding information in schizophrenia, which is linked to dysfunctions in the neural networks underlying attention, manipulation of information, and encoding strategies. Together, our results suggest that all these dysfunctions may be targets for neuromodulation interventions known to improve cognitive deficits in schizophrenia. Copyright © 2017 Elsevier B.V. All rights reserved.

Spectroscopic Investigation of p-Shell Lambda Hypernuclei by the (e,e'K +) Reaction

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

Chen, Chunhua

2014-08-01

Hypernuclear spectroscopy is a powerful tool to investigate Lambda-N interaction. Compared with other Lambda hypernuclei productions, electroproduction via the (e,e'K+) reaction has the advantage of exciting states deeply inside of the hypernucleus and achieving sub-MeV energy resolution. The E05-115 experiment, which was successfully performed in 2009, is the third generation hypernuclear experiment in JLab Hall C. A new splitter magnet and electron spectrometer were installed, and beam energy of 2.344 GeV was selected in this experiment. These new features gave better field uniformity, optics quality and made the tilt method more effective in improving yield-to-background ratio. The magnetic optics ofmore » the spectrometers were carefully studied with GEANT simulation, and corrections were applied to compensate for the fringe field cross talk between the compact spectrometer magnets. The non-linear least chi-squared method was used to further calibrate the spectrometer with the events from Lambda, Sigma0 and B12Lambda and uniform magnetic optics as well as precise kinematics were achieved. Several p-shell Lambda hypernuclear spectra, including B 12 Λ, Be 10 Λ, He 7 Λ, were obtained with high energy resolution and good accuracy. For B 12 Λ, eight peaks were recognized with the resolution of ~540keV (FWHM), and the ground state binding energy was obtained as 11.529 ± 0.012(stat.) ± 0.110(syst.) MeV. Be 10 Λ, twelve peaks were recognized with the resolution of ~520keV (FWHM), and the binding energy of the ground state was determined as 8.710 ± 0.059(stat.) ± 0.114(syst.) MeV. For He 7 Λ, three peaks were recognized with the resolution of ~730keV, and the ground state binding energy was obtained as 5.510 ± 0.050(stat.) ± 0.120(syst.) MeV. Compared with the published data of B 12 Λ from the JLab Hall A experiment, four extra peaks were fitted and interpreted thanks to the highest ever energy resolution and sufficient statistics. The determined binding energy of Be 10 Λ provides new information on charge symmetry breaking effect in the Lambda-N interaction. Compared with the results of He7Lambda from the E01-011 experiment, the ground state position is consistent with 4 times more statistics, and two extra peaks corresponding to excited states were recognized.« less

Strychnine Binding Associated with Glycine Receptors of the Central Nervous System

PubMed Central

Young, Anne B.; Snyder, Solomon H.

1973-01-01

[3H]Strychnine binds to synaptic-membrane fractions of the spinal cord in a selective fashion, indicating an interaction with postsynaptic glycine receptors. Displacement of strychnine by glycine and other amino acids parallels their glycine-like neurophysiologic activity. The regional localization of strychnine binding in the central nervous system correlates closely with endogenous glycine concentrations. In subcellular fractionation experiments, strychnine binding is most enhanced in synaptic-membrane fractions. Strychnine binding is saturable, with affinity constants for glycine and strychnine of 10 and 0.03 μM, respectively. PMID:4200724

Metal loading levels influence on REE distribution on humic acid: Experimental and Modelling approach

NASA Astrophysics Data System (ADS)

Marsac, R.; Davranche, M.; Gruau, G.; Dia, A.

2009-04-01

In natural organic-rich waters, rare earth elements (REE) speciation is mainly controlled by organic colloids such as humic acid (HA). Different series of REE-HA complexation experiments performed at several metal loading (REE/C) displayed two pattern shapes (i) at high metal loading, a middle-REE (MREE) downward concavity, and (ii) at low metal loading, a regular increase from La to Lu (e.g. Sonke and Salters, 2006; Pourret et al., 2007). Both REE patterns might be related to REE binding with different surface sites on HA. To understand REE-HA binding, REE-HA complexation experiments at various metals loading were carried out using ultrafiltration combined with ICP-MS measurements, for the 14 REE simultaneously. The patterns of the apparent coefficients of REE partition between HA and the inorganic solution (log Kd) evolved regularly according to the metal loading. The REE patterns presented a MREE downward concavity at low loading and a regular increase from La to Lu at high loading. The dataset was modelled with Model VI by adjusting two specific parameters, log KMA, the apparent complexation constant of HA low affinity sites and DLK2, the parameter increasing high affinity sites binding strength. Experiments and modelling provided evidence that HA high affinity sites controlled the REE binding with HA at low metal loading. The REE-HA complex could be as multidentate complexes with carboxylic or phenolic sites or potentially with sites constituted of N, P or S as donor atoms. Moreover, these high affinity sites could be different for light and heavy REE, because heavy REE have higher affinity for these sites, in low density, and could saturate them. These new Model VI parameter sets allowed the prediction of the REE-HA pattern shape evolution on a large range of pH and metal loading. According to the metal loading, the evolution of the calculated REE patterns was similar to the various REE pattern observed in natural acidic organic-rich waters (pH<7 and DOC>10 mg L-1). As a consequence, the metal loading could be the key parameter controlling the REE pattern in organic-rich waters.

Shielding voices: The modulation of binding processes between voice features and response features by task representations.

PubMed

Bogon, Johanna; Eisenbarth, Hedwig; Landgraf, Steffen; Dreisbach, Gesine

2017-09-01

Vocal events offer not only semantic-linguistic content but also information about the identity and the emotional-motivational state of the speaker. Furthermore, most vocal events have implications for our actions and therefore include action-related features. But the relevance and irrelevance of vocal features varies from task to task. The present study investigates binding processes for perceptual and action-related features of spoken words and their modulation by the task representation of the listener. Participants reacted with two response keys to eight different words spoken by a male or a female voice (Experiment 1) or spoken by an angry or neutral male voice (Experiment 2). There were two instruction conditions: half of participants learned eight stimulus-response mappings by rote (SR), and half of participants applied a binary task rule (TR). In both experiments, SR instructed participants showed clear evidence for binding processes between voice and response features indicated by an interaction between the irrelevant voice feature and the response. By contrast, as indicated by a three-way interaction with instruction, no such binding was found in the TR instructed group. These results are suggestive of binding and shielding as two adaptive mechanisms that ensure successful communication and action in a dynamic social environment.

Performance of the Cylindrical Drift Chamber and the Inner Plastic Scintillator in the BGOegg experiment

NASA Astrophysics Data System (ADS)

Shibukawa, Takuya; Masumoto, Shinichi; Ozawa, Kyoichiro; Ohnishi, Hiroaki; Muramatsu, Norihito; Ishikawa, Takatsugu; Miyabe, Manabu; Tsuchikawa, Yusuke; Yamazaki, Ryuji; Matsumura, Yuji; Mizutani, Keigo; Hashimoto, Toshikazu; Hamano, Hirotomo; LEPS2/BGOegg Collaboration

2014-09-01

Properties of vector mesons, such as ω mesons, in nucleus are intensively measured to study interactions between mesons and nuclear medium. To study ω meson properties in nuclei, we search for the nuclear ω bound states in the LEPS2/BGOegg experiment at SPring-8. If a strongly bounded ω state exists and binding energy is measured, it gives a phenomenological information about interactions between ω meson and nuclei. ω meson is produced using the GeV γ rays at SPring-8/LEPS2 beamline. The ω bound state is searched from the missing mass measurements of forward going protons. ω meson production is identified by detecting γ and proton from ωN --> N* --> γp or ωN --> γΔ --> γπ p reaction. In the BGOegg experiment, charged particles are detected by Cylindrical Drift Chamber(CDC) and Inner Plastic Scintillators (IPS) around the target. CDC has 4 layers of stereo wires and each layer has 72 sense wires. IPS consists of 30 plastic scintillators. In this talk, the performance of CDC and IPS are described in detail. Properties of vector mesons, such as ω mesons, in nucleus are intensively measured to study interactions between mesons and nuclear medium. To study ω meson properties in nuclei, we search for the nuclear ω bound states in the LEPS2/BGOegg experiment at SPring-8. If a strongly bounded ω state exists and binding energy is measured, it gives a phenomenological information about interactions between ω meson and nuclei. ω meson is produced using the GeV γ rays at SPring-8/LEPS2 beamline. The ω bound state is searched from the missing mass measurements of forward going protons. ω meson production is identified by detecting γ and proton from ωN --> N* --> γp or ωN --> γΔ --> γπ p reaction. In the BGOegg experiment, charged particles are detected by Cylindrical Drift Chamber(CDC) and Inner Plastic Scintillators (IPS) around the target. CDC has 4 layers of stereo wires and each layer has 72 sense wires. IPS consists of 30 plastic scintillators. In this talk, the performance of CDC and IPS are described in detail. All members of the collaboration are listed on http://www.lns.tohoku.ac.jp/ ~bgoegg/collaboration.html

Indistinguishability and identifiability of kinetic models for the MurC reaction in peptidoglycan biosynthesis.

PubMed

Hattersley, J G; Pérez-Velázquez, J; Chappell, M J; Bearup, D; Roper, D; Dowson, C; Bugg, T; Evans, N D

2011-11-01

An important question in Systems Biology is the design of experiments that enable discrimination between two (or more) competing chemical pathway models or biological mechanisms. In this paper analysis is performed between two different models describing the kinetic mechanism of a three-substrate three-product reaction, namely the MurC reaction in the cytoplasmic phase of peptidoglycan biosynthesis. One model involves ordered substrate binding and ordered release of the three products; the competing model also assumes ordered substrate binding, but with fast release of the three products. The two versions are shown to be distinguishable; however, if standard quasi-steady-state assumptions are made distinguishability cannot be determined. Once model structure uniqueness is ensured the experimenter must determine if it is possible to successfully recover rate constant values given the experiment observations, a process known as structural identifiability. Structural identifiability analysis is carried out for both models to determine which of the unknown reaction parameters can be determined uniquely, or otherwise, from the ideal system outputs. This structural analysis forms an integrated step towards the modelling of the full pathway of the cytoplasmic phase of peptidoglycan biosynthesis. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Covert retrieval in working memory impacts the phenomenological characteristics remembered during episodic memory.

PubMed

Loaiza, Vanessa M; Borovanska, Borislava M

2018-01-01

Much research has investigated the qualitative experience of retrieving events from episodic memory (EM). The present study investigated whether covert retrieval in WM increases the phenomenological characteristics that participants find memorable in EM using tasks that distract attention from the maintenance of memoranda (i.e., complex span; Experiment 1) relative to tasks that do not (i.e., short or long list lengths of simple span; Experiments 1 and 2). Participants rated the quality of the phonological, semantic, and temporal-contextual characteristics remembered during a delayed memory characteristics questionnaire (MCQ). Whereas an advantage of the complex over simple span items was observed for each characteristic (Experiment 1), no such difference was observed between short and long trials of simple span (Experiment 2). These results are consistent with the view that covert retrieval in WM promotes content-context bindings that are later accessible from EM for both objective performance and subjective details of the remembered information. Copyright © 2017 Elsevier Inc. All rights reserved.

How Arousal Affects Younger and Older Adults' Memory Binding

PubMed Central

Nashiro, Kaoru; Mather, Mara

2009-01-01

A number of recent studies have shown that associative memory for within-item features is enhanced for emotionally arousing items, whereas arousal-enhanced binding is not seen for associations between distinct items (for a review see Mather, 2007). The costs and benefits of arousal in memory binding have been examined for younger adults but not for older adults. The present experiment examined whether arousal would enhance younger and older adults' within-item and between-item memory binding. The results revealed that arousal improved younger adults' within-item memory binding but not that of older adults. Arousal worsened both groups' between-item memory binding. PMID:21240821

Discover binding pathways using the sliding binding-box docking approach: application to binding pathways of oseltamivir to avian influenza H5N1 neuraminidase

NASA Astrophysics Data System (ADS)

Tran, Diem-Trang T.; Le, Ly T.; Truong, Thanh N.

2013-08-01

Drug binding and unbinding are transient processes which are hardly observed by experiment and difficult to analyze by computational techniques. In this paper, we employed a cost-effective method called "pathway docking" in which molecular docking was used to screen ligand-receptor binding free energy surface to reveal possible paths of ligand approaching protein binding pocket. A case study was applied on oseltamivir, the key drug against influenza a virus. The equilibrium pathways identified by this method are found to be similar to those identified in prior studies using highly expensive computational approaches.

How arousal affects younger and older adults' memory binding.

PubMed

Nashiro, Kaoru; Mather, Mara

2011-01-01

A number of recent studies have shown that associative memory for within-item features is enhanced for emotionally arousing items, whereas arousal-enhanced binding is not seen for associations between distinct items (for a review, see Mather, 2007, Perspectives on Psychological Science, 2, 33-52). The costs and benefits of arousal in memory binding have been examined for younger adults but not for older adults. The present experiment examined whether arousal would enhance younger and older adults' within-item and between-item memory binding. The results revealed that arousal improved younger adults' within-item memory binding but not that of older adults. Arousal worsened both groups' between-item memory binding.

Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.

PubMed

Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica M; Peters, Thomas

2017-07-04

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H, 15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H, 15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[ 13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin.

PubMed

Vijayaraghavan, Balaje; Figueroa, Ricardo A; Bergqvist, Cecilia; Gupta, Amit J; Sousa, Paulo; Hallberg, Einar

2018-06-01

Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane. To investigate the interaction between Samp1 and Emerin in live cells, we performed FRAP experiments in cells overexpressing YFP-Emerin. We compared the mobility of YFP-Emerin in Samp1 knock out cells and cells overexpressing Samp1. The results showed that the mobility of YFP-Emerin was higher in Samp1 knock out cells and lower in cells overexpressing Samp1, suggesting that Samp1 significantly attenuates the mobility of Emerin in the nuclear envelope. FRAP experiments using tsBN2 cells showed that the mobility of Emerin depends on RanGTP. Consistently, in vitro binding experiments showed that the affinity between Samp1 and Emerin is decreased in the presence of Ran, suggesting that Ran attenuates the interaction between Samp1 and Emerin. This is the first demonstration that Ran can regulate the interaction between two proteins in the nuclear envelope. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Pharmacological activities of Vitex agnus-castus extracts in vitro.

PubMed

Meier, B; Berger, D; Hoberg, E; Sticher, O; Schaffner, W

2000-10-01

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species.

PubMed

Udby, Lene; Johnsen, Anders H; Borregaard, Niels

2010-04-01

CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments. We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing. We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG. It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera. Copyright 2009 Elsevier B.V. All rights reserved.

Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein.

PubMed

Gillard, Michel; Chatelain, Pierre; Fuks, Bruno

2006-04-24

A specific binding site for the antiepileptic drug levetiracetam (2S-(oxo-1-pyrrolidinyl)butanamide, Keppra) in rat brain, referred to as the levetiracetam binding site, was discovered several years ago. More recently, this binding site has been identified as the synaptic vesicle protein 2A (SV2A), a protein present in synaptic vesicles [Lynch, B., Lambeng, N., Nocka, K., Kensel-Hammes, P., Bajjalieh, S.M., Matagne, A., Fuks, B., 2004. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc. Natl. Acad. Sci. USA, 101, 9861-9866.]. In this study, we characterized the binding properties of levetiracetam in post-mortem human brain and compared them to human SV2A expressed in Chinese hamster ovary (CHO) cells. The results showed that the binding properties of levetiracetam and [3H]ucb 30889, an analogue that was previously characterized as a suitable ligand for levetiracetam binding site/SV2A in rat brain [Gillard, M., Fuks, B., Michel, P., Vertongen, P., Massingham, R. Chatelain, P., 2003. Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain. Eur. J. Pharmacol. 478, 1-9.], are almost identical in human brain samples (cerebral cortex, hippocampus and cerebellum) and in CHO cell membranes expressing the human SV2A protein. Moreover, the results are also similar to those previously obtained in rat brain. [3H]ucb 30889 binding in human brain and to SV2A was saturable and reversible. At 4 degrees C, its binding kinetics were best fitted assuming a two-phase model in all tissues. The half-times of association for the fast component ranged between 1 to 2 min and represent 30% to 36% of the sites whereas the half-times for the slow component ranged from 20 to 29 min. In dissociation experiments, the half-times were from 2 to 4 min for the fast component (33% to 49% of the sites) and 20 to 41 min for the slow component. Saturation binding curves led to Kd values for [3H]ucb 30889 of 53+/-7, 55+/-9, 70+/-11 and 75+/-33 nM in human cerebral cortex, hippocampus, cerebellum and CHO cells expressing SV2A respectively. Bmax values around 3-4 pmol/mg protein were calculated in all brain regions. Some of the saturation curves displayed curvilinear Scatchard plots indicating the presence of high and low affinity binding sites. When this was the case, Kd values from 25 to 30 nM for the high affinity sites (24% to 34% of total sites) and from 200 to 275 nM for the low affinity sites were calculated. This was observed in all brain regions and in CHO cell membranes expressing the SV2A protein. It cannot be explained by putative binding of [3H]ucb 30889 to SV2B or C isoforms but may reflect different patterns of SV2A glycosylation or the formation of SV2A oligomers. Competition experiments were performed to determine the affinities for SV2A of a variety of compounds including levetiracetam, some of its analogues and other molecules known to interact with levetiracetam binding sites in rat brain such as bemegride, pentylenetetrazol and chlordiazepoxide. We found an excellent correlation between the affinities of these compounds measured in human brain, rat brain and CHO cells expressing human SV2A. In conclusion, we report for the first time that the binding characteristics of native levetiracetam binding sites/SV2A in human brain and rat brain share very similar properties with human recombinant SV2A expressed in CHO cells.

Interactive transport of guanidinylated poly(propylene imine)-based dendrimers through liposomal and cellular membranes.

PubMed

Tsogas, Ioannis; Sideratou, Zili; Tsiourvas, Dimitris; Theodossiou, Theodossis A; Paleos, Constantinos M

2007-10-15

The ability of guanidinylated poly(propylene imine) dendrimers to translocate across lipid bilayers was assessed by employing either a model phosphate-bearing liposomal membrane system or A549 human lung carcinoma cells. Two dendrimer generations, differing in the number of surface guanidinium groups, were employed, while surface acetylation or the use of spacers affected the binding of the guanidinium group to the phosphate moiety and finally the transport efficiency. Following adhesion of dendrimers with liposomes, fusion or transport occurred. Transport through the liposomal bilayer was observed at low guanidinium/phosphate molar ratios, and was enhanced when the bilayer was in the liquid-crystalline phase. For effective transport through the liposomal membrane, an optimum balance between the binding strength and the degree of hydrophobicity of the guanidinylated dendrimer is required. In experiments performed in vitro with cells, efficient penetration and internalization in subcellular organelles and cytosol was observed.

On-chip microlasers for biomolecular detection via highly localized deposition of a multifunctional phospholipid ink.

PubMed

Bog, Uwe; Laue, Thomas; Grossmann, Tobias; Beck, Torsten; Wienhold, Tobias; Richter, Benjamin; Hirtz, Michael; Fuchs, Harald; Kalt, Heinz; Mappes, Timo

2013-07-21

We report on a novel approach to realize on-chip microlasers, by applying highly localized and material-saving surface functionalization of passive photonic whispering gallery mode microresonators. We apply dip-pen nanolithography on a true three-dimensional structure. We coat solely the light-guiding circumference of pre-fabricated poly(methyl methacrylate) resonators with a multifunctional molecular ink. The functionalization is performed in one single fabrication step and simultaneously provides optical gain as well as molecular binding selectivity. This allows for a direct and flexible realization of on-chip microlasers, which can be utilized as biosensors in optofluidic lab-on-a-chip applications. In a proof-of-concept we show how this highly localized molecule deposition suffices for low-threshold lasing in air and water, and demonstrate the capability of the ink-lasers as biosensors in a biotin-streptavidin binding experiment.

Total external reflection X-ray fluorescence analysis of protein-metal ion interactions in biological systems

NASA Astrophysics Data System (ADS)

Novikova, N. N.; Kovalchuk, M. V.; Yur'eva, E. A.; Konovalov, O. V.; Rogachev, A. V.; Stepina, N. D.; Sukhorukov, V. S.; Tsaregorodtsev, A. D.; Chukhrai, E. S.; Yakunin, S. N.

2012-09-01

This paper presents the results of an investigation into hemoglobin-based protein films that were formed on a liquid surface. X-ray standing wave measurements were performed at the ID 10 beamline of the European Synchrotron Radiation Facility (ESRF) and at the Langmuir station of the Kurchatov Synchrotron Radiation Source. It was found that the ability of the protein to bind metal ions is substantially increased due to the conformational rearrangements of protein macromolecules caused by various damaging effects. The elemental composition of protein preparations, which were isolated from children and adults with chronic metabolic diseases accompanied by endogenous intoxication, was analyzed. The results of the investigations offer evidence that an increase in the ligand-binding properties of the protein molecules, which was observed in model experiments using protein films, is a common trait and corresponds to in vivo processes accompanying metabolic disturbances in the body.

Structural characterization of tetranortriterpenes from Pseudrocedrela kotschyi and Trichilia emetica and study of their activity towards the chaperone Hsp90.

PubMed

Piaz, Fabrizio Dal; Malafronte, Nicola; Romano, Adriana; Gallotta, Dario; Belisario, Maria Antonietta; Bifulco, Giuseppe; Gualtieri, Maria Josefine; Sanogo, Rokia; Tommasi, Nunziatina De; Pisano, Claudio

2012-03-01

Investigation of roots extracts Pseudrocedrela kotschyi and Trichilia emetica led to identification of 5 limonoid derivatives, Kotschyins D-H, and 11 known compounds. Their structures were elucidated by extensive 1D and 2D NMR experiments in conjunction with mass spectrometry. A surface plasmon resonance (SPR) approach was adopted to screen their Hsp90 binding capability and kotschyin D showed a significant affinity for the chaperone. Therefore, the characterization of the biological activity of kotschyin D by means of a panel of chemical and biological approaches, including limited proteolysis, molecular docking and biochemical and cellular assays, was performed. Our result indicated this compound as a type of client selective Hsp90 inhibitor, directly binding to the middle domain of the protein and possibly preventing its interaction with the activator of Hsp90 ATPase 1 (Aha1). Copyright © 2011 Elsevier Ltd. All rights reserved.

Narratives of the double bind: Intersectionality in life stories of women of color in physics, astrophysics and astronomy

NASA Astrophysics Data System (ADS)

Ko, Lily T.; Kachchaf, Rachel R.; Ong, Maria; Hodari, Apriel K.

2013-01-01

This paper presents themes on the life stories of women of color in physics, astrophysics and astronomy. Drawing from our NSF-sponsored project, Beyond the Double Bind: Women of Color in STEM, we share findings from 10 interviews and 41 extant texts (about 23 women in varied life stages). Employing intersectionality theory and narrative analysis, our study contributes a critical analysis of how the intersection of gender and race affects performance, identity, persistence and overall career and education experiences in the physical sciences. Our findings both support existing literature on women of color in STEM, as well as bring to light two major, emergent issues: the importance of activism, and school/work-life balance. This research will add to the knowledge base about strategies for retaining women of color--widely considered an untapped source of domestic talent that could fill the country's scientific workforce needs.

Long-Term Pavement Performance Bind Online [Product Brief

DOT National Transportation Integrated Search

2017-02-23

This Product Brief introduces the reader to the Long-Term Pavement Performance Bind (LTPPBind) Online Web-based tool for selecting asphalt binder performance grades (PGs).(1) It explains what the tool is, who can benefit from its use, what its main f...

Cross-modal working memory binding and word recognition skills: how specific is the link?

PubMed

Wang, Shinmin; Allen, Richard J

2018-04-01

Recent research has suggested that the creation of temporary bound representations of information from different sources within working memory uniquely relates to word recognition abilities in school-age children. However, it is unclear to what extent this link is attributable specifically to the binding ability for cross-modal information. This study examined the performance of Grade 3 (8-9 years old) children on binding tasks requiring either temporary association formation of two visual items (i.e., within-modal binding) or pairs of visually presented abstract shapes and auditorily presented nonwords (i.e., cross-modal binding). Children's word recognition skills were related to performance on the cross-modal binding task but not on the within-modal binding task. Further regression models showed that cross-modal binding memory was a significant predictor of word recognition when memory for its constituent elements, general abilities, and crucially, within-modal binding memory were taken into account. These findings may suggest a specific link between the ability to bind information across modalities within working memory and word recognition skills.

Spot Surface Labeling of Magnetic Microbeads and Application in Biological Force Measurements

NASA Astrophysics Data System (ADS)

Estes, Ashley; O'Brien, E. Tim; Hill, David; Superfine, Richard

2006-11-01

Biological force measurements on single molecules and macromolecular structures often use microbeads for the application of force. These techniques are often complicated by multiple attachments and nonspecific binding. In one set of experiments, we are applying a magnetic force microscope that allows us to pull on magnetic beads attached to ciliated human bronchial epithelial cells. These experiments provide a means to measure the stall force of cilia and understand how cilia propel fluids. However, because we are using beads with diameters of one and 2.8 microns, and the diameter of human airway cilia is approximately 200 nm, we cannot be assured that the bead is bound to a single cilium. To address this, we have developed a sputter coating technique to block the biotin binding capability of the streptavidin labeled bead over its entire surface except for a small spot. These beads may also have applications in other biological experiments such as DNA force experiments in which binding of a single target to an individual bead is critical.

Pot binding as a variable confounding plant phenotype: theoretical derivation and experimental observations.

PubMed

Sinclair, Thomas R; Manandhar, Anju; Shekoofa, Avat; Rosas-Anderson, Pablo; Bagherzadi, Laleh; Schoppach, Remy; Sadok, Walid; Rufty, Thomas W

2017-04-01

Theoretical derivation predicted growth retardation due to pot water limitations, i.e., pot binding. Experimental observations were consistent with these limitations. Combined, these results indicate a need for caution in high-throughput screening and phenotyping. Pot experiments are a mainstay in many plant studies, including the current emphasis on developing high-throughput, phenotyping systems. Pot studies can be vulnerable to decreased physiological activity of the plants particularly when pot volume is small, i.e., "pot binding". It is necessary to understand the conditions under which pot binding may exist to avoid the confounding influence of pot binding in interpreting experimental results. In this paper, a derivation is offered that gives well-defined conditions for the occurrence of pot binding based on restricted water availability. These results showed that not only are pot volume and plant size important variables, but the potting media is critical. Artificial potting mixtures used in many studies, including many high-throughput phenotyping systems, are particularly susceptible to the confounding influences of pot binding. Experimental studies for several crop species are presented that clearly show the existence of thresholds of plant leaf area at which various pot sizes and potting media result in the induction of pot binding even though there may be no immediate, visual plant symptoms. The derivation and experimental results showed that pot binding can readily occur in plant experiments if care is not given to have sufficiently large pots, suitable potting media, and maintenance of pot water status. Clear guidelines are provided for avoiding the confounding effects of water-limited pot binding in studying plant phenotype.

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight.

PubMed

Shamsi, Anas; Ahmed, Azaj; Bano, Bilqees

2018-05-01

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 10 4  M -1 implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.

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

PubMed

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

2018-06-01

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

Insight into the coordination and the binding sites of Cu(2+) by the histidyl-6-tag using experimental and computational tools.

PubMed

Watly, Joanna; Simonovsky, Eyal; Wieczorek, Robert; Barbosa, Nuno; Miller, Yifat; Kozlowski, Henryk

2014-07-07

His-tags are specific sequences containing six to nine subsequent histydyl residues, and they are used for purification of recombinant proteins by use of IMAC chromatography. Such polyhistydyl tags, often used in molecular biology, can be also found in nature. Proteins containing histidine-rich domains play a critical role in many life functions in both prokaryote and eukaryote organisms. Binding mode and the thermodynamic properties of the system depend on the specific metal ion and the histidine sequence. Despite the wide application of the His-tag for purification of proteins, little is known about the properties of metal-binding to such tag domains. This inspired us to undertake detailed studies on the coordination of Cu(2+) ion to hexa-His-tag. Experiments were performed using the potentiometric, UV-visible, CD, and EPR techniques. In addition, molecular dynamics (MD) simulations and density functional theory (DFT) calculations were applied. The experimental studies have shown that the Cu(2+) ion binds most likely to two imidazoles and one, two, or three amide nitrogens, depending on the pH. The structures and stabilities of the complexes for the Cu(2+)-Ac-(His)6-NH2 system using experimental and computational tools were established. Polymorphic binding states are suggested, with a possibility of the formation of α-helix structure induced by metal ion coordination. Metal ion is bound to various pairs of imidazole moieties derived from the tag with different efficiencies. The coordination sphere around the metal ion is completed by molecules of water. Finally, the Cu(2+) binding by Ac-(His)6-NH2 is much more efficient compared to other multihistidine protein domains.

The role of ω-subunit of Escherichia coli RNA polymerase in stress response.

PubMed

Bhardwaj, Neerupma; Syal, Kirtimaan; Chatterji, Dipankar

2018-05-01

ppGpp, an alarmone for stringent response, plays an important role in the reprogramming of the transcription complex at the time of stress. In Escherichia coli, ppGpp mediates its action by binding to at least two different sites on RNA polymerase (RNAP). One of the sites to which ppGpp binds to RNAP is at the β'-ω interface; however, the underlying molecular mechanism and the physiological relevance of ppGpp binding to this site remain unclear. In this study, we have performed UV cross-linking experiments using 32 P azido-labeled ppGpp to probe its association with RNAP in the absence and presence of ω, and observed weaker binding of ppGpp to the RNAP without ω. Furthermore, we followed the binding kinetics of ppGpp to RNAP with and without ω by isothermal titration calorimetry and found it to be concurrent with the cross-linking results. Native ω is intrinsically disordered, and we have used a previously characterized structured mutant of ω, which affects the plasticity of the active site of RNAP. Results show that the flexibility conferred by the unstructured ω is a prerequisite for ppGpp binding to RNAP. We have analyzed the stress-associated phenotypes in an E. coli strain devoid of ω (∆rpoZ). ppGpp levels in ∆rpoZ strain were found to be similar to that of the wild-type strain. Interestingly, when the ∆rpoZ strain of E. coli was transferred after nutritional stress to an enriched media, the recovery of growth was compromised. We have identified a new phenotype of ∆rpoZ strain corresponding to defect in biofilm formation in minimal media. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Spectroscopic characterization of the binding mechanism of fluorescein and carboxyfluorescein in human serum albumin

NASA Astrophysics Data System (ADS)

Sulaiman, Saba A. J.; Kulathunga, H. Udani; Abou-Zied, Osama K.

2015-03-01

Fluorescein (FL) and some of its precursors have proven to be effective fluorescent tracers in pharmaceutical and medical applications owing to their high quantum yield of fluorescence in physiological conditions and their high membrane permeability. In order to protect FL from metabolic effects during the process of its delivery, human serum albumin (HSA) has been used as a carrier because of its compatibility with the human body. In the present work, we used spectroscopic methods to characterize the binding mechanisms of FL and one of its derivatives, 5(6)- carboxyfluorescein (CFL), in the HSA protein. The absorbance change of the two ligands (FL and CFL) was quantified as a function of the HSA concentration and the results indicate a moderate binding strength for the two ligands inside HSA (1.00 +/- 0.12 x 104 M-1). The quenching effect of FL(CFL) on the fluorescence intensity of W214 (the sole tryptophan in HSA) indicates that FL and CFL occupy Site I in the protein which is known to bind several hydrophobic drugs. By performing site-competitive experiments, the location of the ligands is determined to be similar to that of the anticoagulant drug warfarin. At higher ratios of [ligand]/[HSA], we observed an upward curvature in the Stern-Volmer plots which indicates that the ligands occupy more pockets in Site I, close to W214. Our results indicate that both ligands bind in HSA with a moderate strength that should not affect their release when used as fluorescent reporters. The chemical and physical identities of the two ligands are also preserved inside the HSA binding sites.

Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies.

PubMed

Fong, Monica; Berrin, Jean-Guy; Paës, Gabriel

2016-01-01

Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher. We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted. Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

Crossing borders to bind proteins--a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set.

PubMed

Baltzer, Lars

2011-06-01

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.

The effect of ageing on recollection: the role of the binding updating process.

PubMed

Boujut, Arnaud; Clarys, David

2016-10-01

The aim of this study was to highlight the underlying process responsible for the age-related deficit in recollection. Through two experiments using the Remember-Know-Guess procedure (Gardiner, J. M., & Richardson-Klavehn, A. [2000]. Remembering and knowing. In The Oxford handbook of memory (pp. 229-244). New York, NY: Oxford University Press) in recognition, we manipulated the opportunity to update bindings between target items and their encoding context, in young and older adults. In the first experiment we impaired the binding updating process during the encoding of items, while in the second we supported this process. The results indicated that the "Remember" responses in the younger group were specifically reduced by the impairment of the binding updating process (Exp. 1), suggesting that this ability is useful for them to encode a specific episode. Conversely, only the "Remember" responses in the older group were improved in accuracy by supporting the binding updating process (Exp. 2), suggesting that their weakness in this ability is the source of their failure to improve the accuracy of their memories. The overall results support the hypothesis that the age-related decline in episodic memory is partly due to a greater vulnerability to interference on bindings, impairing the ability to update content-context bindings as and when events occur.

Molecular conformation of the full-length tumor suppressor NF2/Merlin—a small angle neutron scattering study

PubMed Central

Khajeh, Jahan Ali; Ju, Jeong Ho; Atchiba, Moussoubaou; Allaire, Marc; Stanley, Christopher; Heller, William T.; Callaway, David J.E.; Bu, Zimei

2014-01-01

Summary The tumor suppressor protein Merlin inhibits cell proliferation upon establishing cell-cell contacts. Because Merlin has high sequence similarity to the Ezrin-Radixin-Moesin (ERM) family of proteins, the structural model of ERM protein autoinhibition and cycling between closed/resting and open/active conformational states is often employed to explain Merlin function. However, recent biochemical studies suggest alternative molecular models of Merlin function. Here, we have determined the low resolution molecular structure and binding activity of Merlin and a Merlin(S518D) mutant that mimics the inactivating phosphorylation at S518 using small angle neutron scattering (SANS) and binding experiments. SANS shows that in solution both Merlin and Merlin(S518D) adopt a closed conformation, but binding experiments indicate that a significant fraction of either Merlin or Merlin(S518D) is capable of binding to the target protein NHERF1. Upon binding to the phosphatidylinositol 4,5-bisphosphate lipid, the wild-type Merlin adopts a more open conformation than in solution, but Merlin(S518D) remains in a closed conformation. This study supports a rheostat model of Merlin in NHERF1 binding, and contributes to resolve a controversy about the molecular conformation and binding activity of Merlin. PMID:24882693

SMARTIV: combined sequence and structure de-novo motif discovery for in-vivo RNA binding data.

PubMed

Polishchuk, Maya; Paz, Inbal; Yakhini, Zohar; Mandel-Gutfreund, Yael

2018-05-25

Gene expression regulation is highly dependent on binding of RNA-binding proteins (RBPs) to their RNA targets. Growing evidence supports the notion that both RNA primary sequence and its local secondary structure play a role in specific Protein-RNA recognition and binding. Despite the great advance in high-throughput experimental methods for identifying sequence targets of RBPs, predicting the specific sequence and structure binding preferences of RBPs remains a major challenge. We present a novel webserver, SMARTIV, designed for discovering and visualizing combined RNA sequence and structure motifs from high-throughput RNA-binding data, generated from in-vivo experiments. The uniqueness of SMARTIV is that it predicts motifs from enriched k-mers that combine information from ranked RNA sequences and their predicted secondary structure, obtained using various folding methods. Consequently, SMARTIV generates Position Weight Matrices (PWMs) in a combined sequence and structure alphabet with assigned P-values. SMARTIV concisely represents the sequence and structure motif content as a single graphical logo, which is informative and easy for visual perception. SMARTIV was examined extensively on a variety of high-throughput binding experiments for RBPs from different families, generated from different technologies, showing consistent and accurate results. Finally, SMARTIV is a user-friendly webserver, highly efficient in run-time and freely accessible via http://smartiv.technion.ac.il/.

Age differences in short-term memory binding are related to working memory performance across the lifespan.

PubMed

Fandakova, Yana; Sander, Myriam C; Werkle-Bergner, Markus; Shing, Yee Lee

2014-03-01

Memory performance increases during childhood and adolescence, and decreases in old age. Among younger adults, better ability to bind items to the context in which they were experienced is associated with higher working memory performance (Oberauer, 2005). Here, we examined the extent to which age differences in binding contribute to life span age differences in short-term memory (STM). Younger children (N = 85; 10 to 12 years), teenagers (N = 41; 13 to 15 years), younger adults (N = 84; 20 to 25 years), and older adults (N = 86; 70 to 75 years) worked on global and local short-term recognition tasks that are assumed to measure item and item-context memory, respectively. Structural equation models showed that item-context bindings are functioning less well in children and older adults compared with younger adults and teenagers. This result suggests protracted development of the ability to form and recollect detailed short-term memories, and decline of this ability in aging. Across all age groups, better item-context binding was associated with higher working memory performance, indicating that developmental differences in binding mechanisms are closely related to working memory development in childhood and old age. (c) 2014 APA, all rights reserved.

Functional assignment to JEV proteins using SVM.

PubMed

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

Functional assignment to JEV proteins using SVM

PubMed Central

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

Combining fragment homology modeling with molecular dynamics aims at prediction of Ca2+ binding sites in CaBPs

NASA Astrophysics Data System (ADS)

Pang, ChunLi; Cao, TianGuang; Li, JunWei; Jia, MengWen; Zhang, SuHua; Ren, ShuXi; An, HaiLong; Zhan, Yong

2013-08-01

The family of calcium-binding proteins (CaBPs) consists of dozens of members and contributes to all aspects of the cell's function, from homeostasis to learning and memory. However, the Ca2+-binding mechanism is still unclear for most of CaBPs. To identify the Ca2+-binding sites of CaBPs, this study presented a computational approach which combined the fragment homology modeling with molecular dynamics simulation. For validation, we performed a two-step strategy as follows: first, the approach is used to identify the Ca2+-binding sites of CaBPs, which have the EF-hand Ca2+-binding site and the detailed binding mechanism. To accomplish this, eighteen crystal structures of CaBPs with 49 Ca2+-binding sites are selected to be analyzed including calmodulin. The computational method identified 43 from 49 Ca2+-binding sites. Second, we performed the approach to large-conductance Ca2+-activated K+ (BK) channels which don't have clear Ca2+-binding mechanism. The simulated results are consistent with the experimental data. The computational approach may shed some light on the identification of Ca2+-binding sites in CaBPs.

Assessment of the binding performance of histamine-imprinted microspheres by frontal analysis capillary electrophoresis.

PubMed

Romano, Edwin F; Quirino, Joselito P; Holdsworth, John L; So, Regina C; Holdsworth, Clovia I

2017-05-01

Frontal analysis capillary electrophoresis was used to evaluate the binding performance of molecularly imprinted microspheres (MIM) toward its template histamine and analogs at pH 7, and compared to the high performance liquid chromatographic method. In both methods, batch binding was employed and the binding parameters were calculated from the measured concentration of unbound amine analytes and afforded comparable histamine equilibrium dissociation constants (K d ∼ 0.4 mM). FACE was easily carried out at shorter binding equilibration time (i.e. 30 min) and without the need to separate the microspheres, circumventing laborious and, in the case of the system under study, inefficient sample filtration. It also allowed for competitive binding studies by virtue of its ability to distinctly separate intact microspheres and all tested amines which could not be resolved in HPLC. K d 's for nonimprinted (control) microspheres (NIM) from FACE and HPLC were also comparable (∼ 0.6 mM) but at higher histamine concentrations, HPLC gave lower histamine binding. This discrepancy was attributed to inefficient filtration of the batch binding samples prior to HPLC analysis resulting in an over-estimation of the concentration of free histamine brought about by the presence of unfiltered histamine-bound microspheres. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alchemical Free Energy Calculations for Nucleotide Mutations in Protein-DNA Complexes.

PubMed

Gapsys, Vytautas; de Groot, Bert L

2017-12-12

Nucleotide-sequence-dependent interactions between proteins and DNA are responsible for a wide range of gene regulatory functions. Accurate and generalizable methods to evaluate the strength of protein-DNA binding have long been sought. While numerous computational approaches have been developed, most of them require fitting parameters to experimental data to a certain degree, e.g., machine learning algorithms or knowledge-based statistical potentials. Molecular-dynamics-based free energy calculations offer a robust, system-independent, first-principles-based method to calculate free energy differences upon nucleotide mutation. We present an automated procedure to set up alchemical MD-based calculations to evaluate free energy changes occurring as the result of a nucleotide mutation in DNA. We used these methods to perform a large-scale mutation scan comprising 397 nucleotide mutation cases in 16 protein-DNA complexes. The obtained prediction accuracy reaches 5.6 kJ/mol average unsigned deviation from experiment with a correlation coefficient of 0.57 with respect to the experimentally measured free energies. Overall, the first-principles-based approach performed on par with the molecular modeling approaches Rosetta and FoldX. Subsequently, we utilized the MD-based free energy calculations to construct protein-DNA binding profiles for the zinc finger protein Zif268. The calculation results compare remarkably well with the experimentally determined binding profiles. The software automating the structure and topology setup for alchemical calculations is a part of the pmx package; the utilities have also been made available online at http://pmx.mpibpc.mpg.de/dna_webserver.html .

SPOT-ligand 2: improving structure-based virtual screening by binding-homology search on an expanded structural template library.

PubMed

Litfin, Thomas; Zhou, Yaoqi; Yang, Yuedong

2017-04-15

The high cost of drug discovery motivates the development of accurate virtual screening tools. Binding-homology, which takes advantage of known protein-ligand binding pairs, has emerged as a powerful discrimination technique. In order to exploit all available binding data, modelled structures of ligand-binding sequences may be used to create an expanded structural binding template library. SPOT-Ligand 2 has demonstrated significantly improved screening performance over its previous version by expanding the template library 15 times over the previous one. It also performed better than or similar to other binding-homology approaches on the DUD and DUD-E benchmarks. The server is available online at http://sparks-lab.org . yaoqi.zhou@griffith.edu.au or yuedong.yang@griffith.edu.au. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Screening and investigation of triplex DNA binders from Stephania tetrandra S. Moore by a combination of peak area-fading UHPLC with orbitrap MS and optical spectroscopies.

PubMed

Yang, Hongmei; Wang, Yihan; Yu, Wenjing; Shi, Lei; Wang, Hongfeng; Su, Rui; Chen, Changbao; Liu, Shuying

2018-05-15

The identification and screening of triplex DNA binders are important because these compounds, in many cases, are potential anticancer agents as well as promising drug candidates. Therefore, the ability to screen for these compounds in a high-throughput mode could dramatically improve the drug screening process. A method involving a combination of 96-well plate format and peak area-fading ultra high performance liquid chromatography coupled with Orbitrap mass spectrometry was employed for screening bioactive compounds binding to the triplex DNA from the extracts of Stephania tetrandra S. Moore. Two compounds were screened out and identified as fangchinoline and tetrandrine, based on the comparison of retention time and MS 2 data with those of standards. The binding mechanisms of fangchinoline and tetrandrine at the molecular level were explored using MS 2 , fluorescence spectroscopy, ultraviolet-visible spectroscopy, and circular dichroism. Collision-induced dissociation experiments showed that the complexes with fangchinoline and tetrandrine were dissociated by ligand elimination. According to these measurements, an intercalating binding is the most appropriate binding mode of these two alkaloids to the triplex DNA. The current work provides not only deep insight into alkaloid-triplex DNA complexes but also useful guidelines for the design of efficient anticancer agents. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and β-1,3 glucan-binding protein from Fenneropenaeus indicus.

PubMed

Sivakamavalli, Jeyachandran; Tripathi, Sunil Kumar; Singh, Sanjeev Kumar; Vaseeharan, Baskaralingam

2015-01-01

Lipopolysaccharide and β-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of β-1,3 glucan (β-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

Dispersion- and Exchange-Corrected Density Functional Theory for Sodium Ion Hydration.

PubMed

Soniat, Marielle; Rogers, David M; Rempe, Susan B

2015-07-14

A challenge in density functional theory is developing functionals that simultaneously describe intermolecular electron correlation and electron delocalization. Recent exchange-correlation functionals address those two issues by adding corrections important at long ranges: an atom-centered pairwise dispersion term to account for correlation and a modified long-range component of the electron exchange term to correct for delocalization. Here we investigate how those corrections influence the accuracy of binding free energy predictions for sodium-water clusters. We find that the dual-corrected ωB97X-D functional gives cluster binding energies closest to high-level ab initio methods (CCSD(T)). Binding energy decomposition shows that the ωB97X-D functional predicts the smallest ion-water (pairwise) interaction energy and larger multibody contributions for a four-water cluster than most other functionals - a trend consistent with CCSD(T) results. Also, ωB97X-D produces the smallest amounts of charge transfer and the least polarizable waters of the density functionals studied, which mimics the lower polarizability of CCSD. When compared with experimental binding free energies, however, the exchange-corrected CAM-B3LYP functional performs best (error <1 kcal/mol), possibly because of its parametrization to experimental formation enthalpies. For clusters containing more than four waters, "split-shell" coordination must be considered to obtain accurate free energies in comparison with experiment.

Converging free energies of binding in cucurbit[7]uril and octa-acid host-guest systems from SAMPL4 using expanded ensemble simulations

NASA Astrophysics Data System (ADS)

Monroe, Jacob I.; Shirts, Michael R.

2014-04-01

Molecular containers such as cucurbit[7]uril (CB7) and the octa-acid (OA) host are ideal simplified model test systems for optimizing and analyzing methods for computing free energies of binding intended for use with biologically relevant protein-ligand complexes. To this end, we have performed initially blind free energy calculations to determine the free energies of binding for ligands of both the CB7 and OA hosts. A subset of the selected guest molecules were those included in the SAMPL4 prediction challenge. Using expanded ensemble simulations in the dimension of coupling host-guest intermolecular interactions, we are able to show that our estimates in most cases can be demonstrated to fully converge and that the errors in our estimates are due almost entirely to the assigned force field parameters and the choice of environmental conditions used to model experiment. We confirm the convergence through the use of alternative simulation methodologies and thermodynamic pathways, analyzing sampled conformations, and directly observing changes of the free energy with respect to simulation time. Our results demonstrate the benefits of enhanced sampling of multiple local free energy minima made possible by the use of expanded ensemble molecular dynamics and may indicate the presence of significant problems with current transferable force fields for organic molecules when used for calculating binding affinities, especially in non-protein chemistries.

Converging free energies of binding in cucurbit[7]uril and octa-acid host-guest systems from SAMPL4 using expanded ensemble simulations.

PubMed

Monroe, Jacob I; Shirts, Michael R

2014-04-01

Molecular containers such as cucurbit[7]uril (CB7) and the octa-acid (OA) host are ideal simplified model test systems for optimizing and analyzing methods for computing free energies of binding intended for use with biologically relevant protein-ligand complexes. To this end, we have performed initially blind free energy calculations to determine the free energies of binding for ligands of both the CB7 and OA hosts. A subset of the selected guest molecules were those included in the SAMPL4 prediction challenge. Using expanded ensemble simulations in the dimension of coupling host-guest intermolecular interactions, we are able to show that our estimates in most cases can be demonstrated to fully converge and that the errors in our estimates are due almost entirely to the assigned force field parameters and the choice of environmental conditions used to model experiment. We confirm the convergence through the use of alternative simulation methodologies and thermodynamic pathways, analyzing sampled conformations, and directly observing changes of the free energy with respect to simulation time. Our results demonstrate the benefits of enhanced sampling of multiple local free energy minima made possible by the use of expanded ensemble molecular dynamics and may indicate the presence of significant problems with current transferable force fields for organic molecules when used for calculating binding affinities, especially in non-protein chemistries.

F199E substitution reduced toxicity of Clostridium perfringens epsilon toxin by depriving the receptor binding capability.

PubMed

Kang, Jingjing; Gao, Jie; Yao, Wenwu; Kang, Lin; Gao, Shan; Yang, Hao; Ji, Bin; Li, Ping; Liu, Jing; Yao, Jiahao; Xin, Wenwen; Zhao, Baohua; Wang, Jinglin

2017-07-03

Epsilon toxin (ETX), a potent toxin, is produced by types B and D strains of Clostridium perfringens, which could cause severe diseases in humans and domestic animals. Mutant rETX F199E was previously demonstrated to be a good vaccine candidate. However, the mechanism concerned remains unknown. To clarify how F199E substitution reduced ETX toxicity, we performed a series of experiments. The results showed that the cell-binding and pore-forming ability of rETX F199E was almost abolished. We speculated that F199E substitution reduced toxicity by depriving the receptor binding capability of ETX, which contributed to the hypothesis that domain I of ETX is responsible for cell binding. In addition, our data suggested that ETX could cause Ca 2+ release from intracellular Ca 2+ stores, which may underlie an alternate pathway leading to cell death. Furthermore, ETX induced crenation of the MDCK cells was observed, with sags and crests first appearing on the surface of condensed MDCK cells, according to scanning electron microscopy. The data also demonstrated the safety and potentiality of rETX F199E as a vaccine candidate for humans. In summary, findings of this work potentially contribute to a better understanding of the pathogenic mechanism of ETX and the development of vaccine against diseases caused by ETX, using mutant proteins.

F199E substitution reduced toxicity of Clostridium perfringens epsilon toxin by depriving the receptor binding capability

PubMed Central

Kang, Jingjing; Gao, Jie; Yao, Wenwu; Kang, Lin; Gao, Shan; Yang, Hao; Ji, Bin; Li, Ping; Liu, Jing; Yao, Jiahao; Xin, Wenwen; Zhao, Baohua; Wang, Jinglin

2017-01-01

ABSTRACT Epsilon toxin (ETX), a potent toxin, is produced by types B and D strains of Clostridium perfringens, which could cause severe diseases in humans and domestic animals. Mutant rETXF199E was previously demonstrated to be a good vaccine candidate. However, the mechanism concerned remains unknown. To clarify how F199E substitution reduced ETX toxicity, we performed a series of experiments. The results showed that the cell-binding and pore-forming ability of rETXF199E was almost abolished. We speculated that F199E substitution reduced toxicity by depriving the receptor binding capability of ETX, which contributed to the hypothesis that domain I of ETX is responsible for cell binding. In addition, our data suggested that ETX could cause Ca2+ release from intracellular Ca2+ stores, which may underlie an alternate pathway leading to cell death. Furthermore, ETX induced crenation of the MDCK cells was observed, with sags and crests first appearing on the surface of condensed MDCK cells, according to scanning electron microscopy. The data also demonstrated the safety and potentiality of rETXF199E as a vaccine candidate for humans. In summary, findings of this work potentially contribute to a better understanding of the pathogenic mechanism of ETX and the development of vaccine against diseases caused by ETX, using mutant proteins. PMID:28304231

Cell membrane antigen-antibody complex dissociation by the widely used glycine-HCL method: an unreliable procedure for studying antibody internalization.

PubMed

Tsaltas, G; Ford, C H

1993-02-01

Methods following the process of binding and internalization of antibodies to cell surface antigens have often employed low pH isoosmolar buffers in order to dissociate surface antigen-antibody complexes. One of the most widely used buffers is a 0.05 M glycine-HCL buffer pH 2.8. Since the efficacy of action of this buffer was critical to a series of internalization experiments employing monoclonal antibodies (Mabs) to carcinoembryonic antigen (CEA) expressing cancer cell lines in this laboratory, we tested its performance in a number of different assays. Our results indicate that this buffer only partially dissociates antigen-antibody bonds and therefore can introduce major inaccuracies in internalization experiments.

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein.

PubMed

Eberini, Ivano; Guerini Rocco, Alessandro; Ientile, Anna Rita; Baptista, António M; Gianazza, Elisabetta; Tomaselli, Simona; Molinari, Henriette; Ragona, Laura

2008-06-01

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family. (c) 2008 Wiley-Liss, Inc.

Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

PubMed

Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

2013-01-01

ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins.

Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70

PubMed Central

Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

2013-01-01

ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins. PMID:24348227

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

ERIC Educational Resources Information Center

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

1976-01-01

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

Effects of Emotion on Associative Recognition: Valence and Retention Interval Matter

PubMed Central

Pierce, Benton H.; Kensinger, Elizabeth A.

2011-01-01

In two experiments, we examined the effects of emotional valence and arousal on associative binding. Participants studied negative, positive, and neutral word pairs, followed by an associative recognition test. In Experiment 1, with a short-delayed test, accuracy for intact pairs was equivalent across valences, whereas accuracy for rearranged pairs was lower for negative than for positive and neutral pairs. In Experiment 2, we tested participants after a one-week delay and found that accuracy was greater for intact negative than for intact neutral pairs, whereas rearranged pair accuracy was equivalent across valences. These results suggest that, although negative emotional valence impairs associative binding after a short delay, it may improve binding after a longer delay. The results also suggest that valence, as well as arousal, needs to be considered when examining the effects of emotion on associative memory. PMID:21401233

Self-Organization of Metal Nanoparticles in Light: Electrodynamics-Molecular Dynamics Simulations and Optical Binding Experiments.

PubMed

McCormack, Patrick; Han, Fei; Yan, Zijie

2018-02-01

Light-driven self-organization of metal nanoparticles (NPs) can lead to unique optical matter systems, yet simulation of such self-organization (i.e., optical binding) is a complex computational problem that increases nonlinearly with system size. Here we show that a combined electrodynamics-molecular dynamics simulation technique can simulate the trajectories and predict stable configurations of silver NPs in optical fields. The simulated dynamic equilibrium of a two-NP system matches the probability density of oscillations for two optically bound NPs obtained experimentally. The predicted stable configurations for up to eight NPs are further compared to experimental observations of silver NP clusters formed by optical binding in a Bessel beam. All configurations are confirmed to form in real systems, including pentagonal clusters with five-fold symmetry. Our combined simulations and experiments have revealed a diverse optical matter system formed by anisotropic optical binding interactions, providing a new strategy to discover artificial materials.

Arousal-Enhanced Location Memory for Pictures

PubMed Central

Mather, Mara; Nesmith, Kathryn

2008-01-01

Four experiments revealed arousal-enhanced location memory for pictures. After an incidental encoding task, participants were more likely to remember the locations of positive and negative arousing pictures than the locations of non-arousing pictures, indicating better binding of location to picture. This arousal-enhanced binding effect did not have a cost for the binding of nearby pictures to their locations. Thus, arousal can enhance binding of an arousing picture’s content to its location without interfering with picture-location binding for nearby pictures. In addition, arousal-enhanced picture-location memory binding is not just a side effect of enhanced memory for the picture itself, as it occurs both when recognition memory is good and when it is poor. PMID:19190722

Principal component analysis on molecular descriptors as an alternative point of view in the search of new Hsp90 inhibitors.

PubMed

Lauria, Antonino; Ippolito, Mario; Almerico, Anna Maria

2009-10-01

Inhibiting a protein that regulates multiple signal transduction pathways in cancer cells is an attractive goal for cancer therapy. Heat shock protein 90 (Hsp90) is one of the most promising molecular targets for such an approach. In fact, Hsp90 is a ubiquitous molecular chaperone protein that is involved in folding, activating and assembling of many key mediators of signal transduction, cellular growth, differentiation, stress-response and apoptothic pathways. With the aim to analyze which molecular descriptors have the higher importance in the binding interactions of these classes, we first performed molecular docking experiments on the 187 Hsp90 inhibitors included in the BindingDB, a public database of measured binding affinities. Further, for each frozen conformation obtained from the docking, a set of 250 molecular descriptors was calculated, and the resulting Structure/Descriptors matrix was submitted to Principal Component Analysis. From the factor scores it emerged a good clusterization among similar compounds both in terms of structural class and activity spectrum, while examination of the loadings of the first two factors also allowed to study the classes of descriptors which mainly contribute to each one.

1-(2-aminophenyl)-1H-1,2,3-triazole-4-carboxylic acid: activity against Gram-positive and Gram-negative pathogens including Vibrio cholerae

NASA Astrophysics Data System (ADS)

Maji, Krishnendu; Haldar, Debasish

2017-10-01

We report a new synthetic aromatic ε-amino acid containing a triazole moiety with antimicrobial potential against Gram-positive, Gram-negative and pathogenic bacteria including Vibrio cholerae. Structure-property relationship studies revealed that all the functional groups are essential to enhance the antimicrobial activity. The 1-(2-aminophenyl)-1H-1,2,3-triazole-4-carboxylic acid was synthesized by click chemistry. From X-ray crystallography, the amino acid adopts a kink-like structure where the phenyl and triazole rings are perpendicular to each other and the amine and acid groups maintain an angle of 60°. The agar diffusion test shows that the amino acid has significant antibacterial activity. The liquid culture test exhibits that the minimum inhibitory concentration (MIC) value for Bacillus subtilis and Vibrio cholerae is 59.5 µg ml-1. FE-SEM experiments were performed to study the morphological changes of bacterial shape after treatment with compound 1. The antimicrobial activity of the amino acid was further studied by DNA binding and degradation study, protein binding, dye-binding assay and morphological analysis. Moreover, the amino acid does not have any harmful effect on eukaryotes.

Interaction between holo transferrin and HSA-PPIX complex in the presence of lomefloxacin: An evaluation of PPIX aggregation in protein-protein interactions

NASA Astrophysics Data System (ADS)

Sattar, Zohreh; Iranfar, Hediye; Asoodeh, Ahmad; Saberi, Mohammad Reza; Mazhari, Mahboobeh; Chamani, Jamshidkhan

2012-11-01

Human serum albumin (HSA) and holo transferrin (TF) are two serum carrier proteins that are able to interact with each other, thereby altering their binding behavior toward their ligands. During the course of this study, the interaction between HSA-PPIX and TF, in the presence and absence of lomefloxacin (LMF), was for the first time investigated using different spectroscopic and molecular modeling techniques. Fluorescence spectroscopy experiments were performed in order to study conformational changes of proteins. The RLS technique was utilized to investigate the effect of LMF on J-aggregation of PPIX, which is the first report of its kind. Our findings present clear-cut evidence for the alteration of interactions between HSA and TF in the presence of PPIX and changes in drug-binding to HSA and HSA-PPIX complex upon interaction with TF. Moreover, molecular modeling studies suggested that the binding site for LMF became switched in the presence of PPIX, and that LMF bound to the site IIA of HSA. The obtained results should give new insight into research in this field and may cast some light on the dynamics of drugs in biological systems.

The heparin-Ca(2+) interaction: the influence of the O-sulfation pattern on binding.

PubMed

Chevalier, Franck; Lucas, Ricardo; Angulo, Jesús; Martin-Lomas, Manuel; Nieto, Pedro M

2004-04-02

The specific binding of Ca(2+) to synthetic hexasaccharide models of modified heparin has been investigated by NMR and molecular modeling and compared with previous results on a model of regular heparin. These two models represent the regular region of heparin lacking one type of O-sulfate group, either at C-6 of glucosamine or at C-2 of iduronate. The NMR experiments show different responses to the presence of Ca(2+). In the case of the compound lacking O-sulfate groups at C-2, the results are indicative of specific binding similar to that observed for the regular heparin, while the model lacking sulfate groups in position 6 interacts more weakly with Ca(2+). In order to understand the basis of this difference, a molecular modeling study based on a rigid body docking approach of the interaction of these carbohydrates with Ca(2+) and Na(+) was performed. We have found that the results are strongly dependent on the starting orientation of the lateral side chains of the charged groups of the carbohydrate, and that the best agreement with the experimental results is obtained when the starting conformations are taken from previous simulations in the presence of Ca(2+).

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

PubMed

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

2016-06-01

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

Ageing and feature binding in visual working memory: The role of presentation time.

PubMed

Rhodes, Stephen; Parra, Mario A; Logie, Robert H

2016-01-01

A large body of research has clearly demonstrated that healthy ageing is accompanied by an associative memory deficit. Older adults exhibit disproportionately poor performance on memory tasks requiring the retention of associations between items (e.g., pairs of unrelated words). In contrast to this robust deficit, older adults' ability to form and temporarily hold bound representations of an object's surface features, such as colour and shape, appears to be relatively well preserved. However, the findings of one set of experiments suggest that older adults may struggle to form temporary bound representations in visual working memory when given more time to study objects. However, these findings were based on between-participant comparisons across experimental paradigms. The present study directly assesses the role of presentation time in the ability of younger and older adults to bind shape and colour in visual working memory using a within-participant design. We report new evidence that giving older adults longer to study memory objects does not differentially affect their immediate memory for feature combinations relative to individual features. This is in line with a growing body of research suggesting that there is no age-related impairment in immediate memory for colour-shape binding.

Osteogenic properties of a short BMP-2 chimera peptide.

PubMed

Falcigno, Lucia; D'Auria, Gabriella; Calvanese, Luisa; Marasco, Daniela; Iacobelli, Roberta; Scognamiglio, Pasqualina L; Brun, Paola; Danesin, Roberta; Pasqualin, Matteo; Castagliuolo, Ignazio; Dettin, Monica

2015-09-01

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP-2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP-2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide-receptor binding interface were highlighted by docking experiments. Peptide-receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73-92 and 30-34 of BMP-2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP-2. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

"Life-span development of visual working memory: When is feature binding difficult?": Correction to Cowan et al. (2006).

PubMed

2016-10-01

Reports an error in "Life-span development of visual working memory: When is feature binding difficult?" by Nelson Cowan, Moshe Naveh-Benjamin, Angela Kilb and J. Scott Saults ( Developmental Psychology , 2006[Nov], Vol 42[6], 1089-1102). In the article, there were two errors in experiment 1a. The mean for color item information in older adults was incorrectly calculated. As a result, Figure 3 shows a mean of over .70. The true mean was .63 ( SEM =.04). This change diminishes the magnitude of the aging deficit for associative information, although this deficit still appears to remain, to a smaller extent. (For a conceptual replication see Peterson & Naveh-Benjamin, 2016). There also was an error in the experimental procedure of Experiment 1a. The older adults in that experiment received only half the number of trials specified in the methods section, and half as much as the other groups. For all groups, when there were 4 or 6 items and the probe was a binding change, the probed location was matched by the same color at 1 other location but, when there were 8 or 10 squares, the probed location was matched by the same color at 1, 2, or 3 other locations. For 8 squares the number of trials was identical for these three trial subtypes whereas, for 10 squares, most of the trials had the same color at just 1 other location. These errors suggest that the experiment should be taken as only preliminary evidence that there is an aging deficit in color-location binding in visual working memory when color and binding trials are mixed in the same trial blocks. (The following abstract of the original article appeared in record 2006-20488-009.) We asked whether the ability to keep in working memory the binding between a visual object and its spatial location changes with development across the life span more than memory for item information. Paired arrays of colored squares were identical or differed in the color of one square, and in the latter case, the changed color was unique on that trial (item change) or was duplicated elsewhere in the array (color-location binding change). Children (8-10 and 11-12 years old) and older adults (65-85 years old) showed deficits relative to young adults. These were only partly simulated by dividing attention in young adults. The older adults had an additional deficiency, specifically in binding information, which was evident only when item- and binding-change trials were mixed together. In that situation, the older adults often overlooked the more subtle, binding-type changes. Some working memory processes related to binding undergo life-span development in an inverted-U shape, whereas other, bias- and salience-related processes that influence the use of binding information seem to develop monotonically. PsycINFO Database Record (c) 2016 APA, all rights reserved

Predicting protein-binding regions in RNA using nucleotide profiles and compositions.

PubMed

Choi, Daesik; Park, Byungkyu; Chae, Hanju; Lee, Wook; Han, Kyungsook

2017-03-14

Motivated by the increased amount of data on protein-RNA interactions and the availability of complete genome sequences of several organisms, many computational methods have been proposed to predict binding sites in protein-RNA interactions. However, most computational methods are limited to finding RNA-binding sites in proteins instead of protein-binding sites in RNAs. Predicting protein-binding sites in RNA is more challenging than predicting RNA-binding sites in proteins. Recent computational methods for finding protein-binding sites in RNAs have several drawbacks for practical use. We developed a new support vector machine (SVM) model for predicting protein-binding regions in mRNA sequences. The model uses sequence profiles constructed from log-odds scores of mono- and di-nucleotides and nucleotide compositions. The model was evaluated by standard 10-fold cross validation, leave-one-protein-out (LOPO) cross validation and independent testing. Since actual mRNA sequences have more non-binding regions than protein-binding regions, we tested the model on several datasets with different ratios of protein-binding regions to non-binding regions. The best performance of the model was obtained in a balanced dataset of positive and negative instances. 10-fold cross validation with a balanced dataset achieved a sensitivity of 91.6%, a specificity of 92.4%, an accuracy of 92.0%, a positive predictive value (PPV) of 91.7%, a negative predictive value (NPV) of 92.3% and a Matthews correlation coefficient (MCC) of 0.840. LOPO cross validation showed a lower performance than the 10-fold cross validation, but the performance remains high (87.6% accuracy and 0.752 MCC). In testing the model on independent datasets, it achieved an accuracy of 82.2% and an MCC of 0.656. Testing of our model and other state-of-the-art methods on a same dataset showed that our model is better than the others. Sequence profiles of log-odds scores of mono- and di-nucleotides were much more powerful features than nucleotide compositions in finding protein-binding regions in RNA sequences. But, a slight performance gain was obtained when using the sequence profiles along with nucleotide compositions. These are preliminary results of ongoing research, but demonstrate the potential of our approach as a powerful predictor of protein-binding regions in RNA. The program and supporting data are available at http://bclab.inha.ac.kr/RBPbinding .

The RNA Hydrolysis and the Cytokinin Binding Activities of PR-10 Proteins Are Differently Performed by Two Isoforms of the Pru p 1 Peach Major Allergen and Are Possibly Functionally Related[W

PubMed Central

Zubini, Paola; Zambelli, Barbara; Musiani, Francesco; Ciurli, Stefano; Bertolini, Paolo; Baraldi, Elena

2009-01-01

PR-10 proteins are a family of pathogenesis-related (PR) allergenic proteins playing multifunctional roles. The peach (Prunus persica) major allergen, Pru p 1.01, and its isoform, Pru p 1.06D, were found highly expressed in the fruit skin at the pit hardening stage, when fruits transiently lose their susceptibility to the fungal pathogen Monilinia spp. To investigate the possible role of the two Pru p 1 isoforms in plant defense, the recombinant proteins were expressed in Escherichia coli and purified. Light scattering experiments and circular dichroism spectroscopy showed that both proteins are monomers in solution with secondary structures typical of PR-10 proteins. Even though the proteins do not display direct antimicrobial activity, they both act as RNases, a function possibly related to defense. The RNase activity is different for the two proteins, and only that of Pru p 1.01 is affected in the presence of the cytokinin zeatin, suggesting a physiological correlation between Pru p 1.01 ligand binding and enzymatic activity. The binding of zeatin to Pru p 1.01 was evaluated using isothermal titration calorimetry, which provided information on the stoichiometry and on the thermodynamic parameters of the interaction. The structural architecture of Pru p 1.01 and Pru p 1.06D was obtained by homology modeling, and the differences in the binding pockets, possibly accounting for the observed difference in binding activity, were evaluated. PMID:19474212

Role of Annular Lipids in the Functional Properties of Leucine Transporter LeuT Proteomicelles.

PubMed

LeVine, Michael V; Khelashvili, George; Shi, Lei; Quick, Matthias; Javitch, Jonathan A; Weinstein, Harel

2016-02-16

Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl β-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins.

Role of Annular Lipids in the Functional Properties of Leucine Transporter LeuT Proteomicelles

PubMed Central

2016-01-01

Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl β-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins. PMID:26811944

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

PubMed Central

Isa, P; López, S; Segovia, L; Arias, C F

1997-01-01

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8. PMID:9261399

A novel injection strategy of flurbiprofen axetil by inhibiting protein binding with 6-methoxy-2-naphthylacetic acid.

PubMed

Ogata, Kenji; Takamura, Norito; Tokunaga, Jin; Ikeda, Tetsuya; Setoguchi, Nao; Tanda, Kazuhiro; Yamasaki, Tetsuo; Nishio, Toyotaka; Kawai, Keiichi

2016-04-01

Flurbiprofen axetil (FPA) is an injection product and a prodrug of a non-steroidal anti-inflammatory drug (NSAID). After injection, it is rapidly hydrolyzed to the active form, flurbiprofen (FP). Since frequent injections of FPA can lead to abnormal physiology, an administration strategy is necessary to ensure there is enhancement of the analgesic efficiency of FP after a single dose and to reduce the total number of doses. FP strongly binds to site II of albumin, and thus the free (unbound) FP concentration is low. This study focused on 6-methoxy-2-naphthylacetic acid (6-MNA), the active metabolite of nabumetone (a prodrug of NSAID). We performed ultrafiltration experiments and pharmacokinetics analysis in rats to investigate whether the inhibitory effect of 6-MNA on FP binding to albumin increased the free FP concentration in vitro and in vivo. Results indicated that 6-MNA inhibited the binding of FP to albumin competitively. When 6-MNA was injected in rats, there was a significant increase in the free FP concentration and the area under concentration-time curve (AUC) calculated from the free FP concentration, while there was a significant decrease in the total (bound + free) FP concentration and the AUC calculated from the total FP concentration. These findings indicate that 6-MNA inhibits the protein binding of FP in vivo. This suggests that the frequency of FPA injections can be reduced when administered with nabumetone, as there is increase in the free FP concentration associated with pharmacological effect.

Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29

PubMed Central

Xu, Yiren; Oruganti, Sri Vidya; Gopalan, Venkat; Foster, Mark P.

2014-01-01

We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg2+-dependent cleavage of the 5′ leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5-RPP30 and RPP21-RPP29. NMR studies of the Pyrococcus furiosus RPP21 and RPP29 proteins in their free and complexed states provided evidence for significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, pH values and in buffers with varying ionization potential, and with a folding-deficient RPP21 point mutant. These experiments revealed a negative heat capacity change (ΔCp), nearly twice that predicted from surface accessibility calculations, a strong salt dependence to the interaction and proton release at neutral pH, but a small net contribution from these to the excess ΔCp. We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess ΔCp. These findings provide novel structural-thermodynamic insights into coupled equilibria that enable specificity in macromolecular assemblies. PMID:22243443

Deuterium Labeling Together with Contrast Variation Small-angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins

PubMed Central

Zaccai, Nathan R.; Sandlin, Clifford W.; Hoopes, James T.; Curtis, Joseph E.; Fleming, Patrick J.; Fleming, Karen G.; Krueger, Susan

2016-01-01

In gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archeal prefoldins and the mitochondrial Tim chaperones, that is α-helical ‘tentacles’ extend from a β-strand ‘body’ to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis and structure modeling. PMID:26791979

Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae

PubMed Central

Chatterjee, Tanaya; Sheikh, Irshad Ali; Chakravarty, Devlina; Chakrabarti, Pinak; Sarkar, Paramita; Saha, Tultul; Chakrabarti, Manoj K.; Hoque, Kazi Mirajul

2015-01-01

Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders. PMID:26540279

Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins.

PubMed

Zaccai, Nathan R; Sandlin, Clifford W; Hoopes, James T; Curtis, Joseph E; Fleming, Patrick J; Fleming, Karen G; Krueger, Susan

2016-01-01

In Gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archaeal prefoldins and the mitochondrial Tim chaperones, that is the α-helical "tentacles" extend from a β-strand "body" to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis, and structure modeling. © 2016 Elsevier Inc. All rights reserved.

Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha sub 1 -adrenergic system

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

Han, H.M.

1989-01-01

During development, the cardiac alpha{sub 1}-adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha{sub 1}-adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha{sub 1}-selective radioligand ({sup 125}I)-I-2-({beta}-(4-hydroxphenyl)ethylaminomethyl)tetralone (({sup 125}I)-IBE 2254) demonstrated that the alpha{sub 1}-adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha{sub 1}-adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha{sub 1}-adrenergic receptor is coupled to both a PT-insensitivemore » and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha{sub 1}-mediated positive chronotropic response in the neonate, whereas in the adult the alpha{sub 1}-mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha{sub 1}-adrenergic receptor to its effector under certain circumstances such as high potassium (K{sup +}) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha{sub 1}-response.« less

Development and validation of an LC-ESI-MS/MS method for the quantification of D-84, reboxetine and citalopram for their use in MS Binding Assays addressing the monoamine transporters hDAT, hSERT and hNET.

PubMed

Neiens, Patrick; De Simone, Angela; Ramershoven, Anna; Höfner, Georg; Allmendinger, Lars; Wanner, Klaus T

2018-03-03

MS Binding Assays represent a label-free alternative to radioligand binding assays. In this study, we present an LC-ESI-MS/MS method for the quantification of (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol [(R,R)-D-84, (R,R)-1], (S,S)-reboxetine [(S,S)-2], and (S)-citalopram [(S)-3] employed as highly selective nonlabeled reporter ligands in MS Binding Assays addressing the dopamine [DAT, (R,R)-D-84], norepinephrine [NET, (S,S)-reboxetine] and serotonin transporter [SERT, (S)-citalopram], respectively. The developed LC-ESI-MS/MS method uses a pentafluorphenyl stationary phase in combination with a mobile phase composed of acetonitrile and ammonium formate buffer for chromatography and a triple quadrupole mass spectrometer in the multiple reaction monitoring mode for mass spectrometric detection. Quantification is based on deuterated derivatives of all three analytes serving as internal standards. The established LC-ESI-MS/MS method enables fast, robust, selective and highly sensitive quantification of all three reporter ligands in a single chromatographic run. The method was validated according to the Center for Drug Evaluation and Research (CDER) guideline for bioanalytical method validation regarding selectivity, accuracy, precision, calibration curve and sensitivity. Finally, filtration-based MS Binding Assays were performed for all three monoamine transporters based on this LC-ESI-MS/MS quantification method as read out. The affinities determined in saturation experiments for (R,R)-D-84 toward hDAT, for (S,S)-reboxetine toward hNET, and for (S)-citalopram toward hSERT, respectively, were in good accordance with results from literature, clearly demonstrating that the established MS Binding Assays have the potential to be an efficient alternative to radioligand binding assays widely used for this purpose so far. Copyright © 2018 John Wiley & Sons, Ltd.

The conserved potassium channel filter can have distinct ion binding profiles: Structural analysis of rubidium, cesium, and barium binding in NaK2K

PubMed Central

Lam, Yee Ling; Zeng, Weizhong; Sauer, David Bryant

2014-01-01

Potassium channels are highly selective for K+ over the smaller Na+. Intriguingly, they are permeable to larger monovalent cations such as Rb+ and Cs+ but are specifically blocked by the similarly sized Ba2+. In this study, we used structural analysis to determine the binding profiles for these permeant and blocking ions in the selectivity filter of the potassium-selective NaK channel mutant NaK2K and also performed permeation experiments using single-channel recordings. Our data revealed that some ion binding properties of NaK2K are distinct from those of the canonical K+ channels KcsA and MthK. Rb+ bound at sites 1, 3, and 4 in NaK2K, as it does in KcsA. Cs+, however, bound predominantly at sites 1 and 3 in NaK2K, whereas it binds at sites 1, 3, and 4 in KcsA. Moreover, Ba2+ binding in NaK2K was distinct from that which has been observed in KcsA and MthK, even though all of these channels show similar Ba2+ block. In the presence of K+, Ba2+ bound to the NaK2K channel at site 3 in conjunction with a K+ at site 1; this led to a prolonged block of the channel (the external K+-dependent Ba2+ lock-in state). In the absence of K+, however, Ba2+ acts as a permeating blocker. We found that, under these conditions, Ba2+ bound at sites 1 or 0 as well as site 3, allowing it to enter the filter from the intracellular side and exit from the extracellular side. The difference in the Ba2+ binding profile in the presence and absence of K+ thus provides a structural explanation for the short and prolonged Ba2+ block observed in NaK2K. PMID:25024267

The conserved potassium channel filter can have distinct ion binding profiles: structural analysis of rubidium, cesium, and barium binding in NaK2K.

PubMed

Lam, Yee Ling; Zeng, Weizhong; Sauer, David Bryant; Jiang, Youxing

2014-08-01

Potassium channels are highly selective for K(+) over the smaller Na(+). Intriguingly, they are permeable to larger monovalent cations such as Rb(+) and Cs(+) but are specifically blocked by the similarly sized Ba(2+). In this study, we used structural analysis to determine the binding profiles for these permeant and blocking ions in the selectivity filter of the potassium-selective NaK channel mutant NaK2K and also performed permeation experiments using single-channel recordings. Our data revealed that some ion binding properties of NaK2K are distinct from those of the canonical K(+) channels KcsA and MthK. Rb(+) bound at sites 1, 3, and 4 in NaK2K, as it does in KcsA. Cs(+), however, bound predominantly at sites 1 and 3 in NaK2K, whereas it binds at sites 1, 3, and 4 in KcsA. Moreover, Ba(2+) binding in NaK2K was distinct from that which has been observed in KcsA and MthK, even though all of these channels show similar Ba(2+) block. In the presence of K(+), Ba(2+) bound to the NaK2K channel at site 3 in conjunction with a K(+) at site 1; this led to a prolonged block of the channel (the external K(+)-dependent Ba(2+) lock-in state). In the absence of K(+), however, Ba(2+) acts as a permeating blocker. We found that, under these conditions, Ba(2+) bound at sites 1 or 0 as well as site 3, allowing it to enter the filter from the intracellular side and exit from the extracellular side. The difference in the Ba(2+) binding profile in the presence and absence of K(+) thus provides a structural explanation for the short and prolonged Ba(2+) block observed in NaK2K. © 2014 Lam et al.

Adherence of oral streptococci: evidence for nonspecific adsorption to saliva-coated hydroxylapatite surfaces.

PubMed Central

Staat, R H; Peyton, J C

1984-01-01

It is proposed that binding of oral streptococci to saliva-coated hydroxylapatite (SHA) surfaces is a multifactorial process involving both specific and nonspecific receptors. In this context, specific binding is described as a high-affinity, saturable interaction between the cell and binding surface. Conversely, nonspecific binding is considered to be a nonsaturable, generalized, low-affinity reaction. Experimental differentiation of specific binding from nonspecific binding was achieved with a competition assay which utilized a large excess of nonradiolabeled bacteria to compete with the 3H-labeled cells for attachment to receptors on 1.5 mg of SHA crystals. Competition assays of Streptococcus sanguis and Streptococcus mitis adhesion clearly demonstrated that the total binding isotherm was composed of a saturable specific binding reaction and a minor nonspecific binding component. This was further substantiated by analysis of nonlinear Scatchard plots of the total binding data. The competition data for Streptococcus mutans binding indicated that ca. 50% of the S. mutans binding appeared to be specific, although saturation of the SHA surfaces with bacterial cells could not be demonstrated. Experiments measuring desorption of radiolabeled cells from SHA crystals into buffer showed that ca. 50% of the bound S. mutans cells were removed after 4 h, whereas less than 5% of the S. sanguis cells were eluted from the SHA surfaces. The kinetics of attachment were studied by using an extract of Persea americana as a noncompetitive inhibitor of adherence. The total cell binding data for these experiments suggested a very rapid binding reaction followed by a slower rate of attachment. It was concluded from these three different experimental approaches that adherence of selected oral streptococci to SHA surfaces involves specific, high-affinity and nonspecific, low-affinity binding reactions. The concept is developed that in vitro streptococcal attachment to SHA can be described as a two-reaction process in which the low-affinity interaction of the cell with the SHA surface precedes the establishment of the stronger, specific bonds needed for the maintenance of streptococci in the oral cavity. PMID:6327530

SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor

PubMed Central

Bradley, ME; Bond, ME; Manini, J; Brown, Z; Charlton, SJ

2009-01-01

Background and purpose: In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric. Experimental approach: To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [35S]-GTPγS binding approaches in addition to chemotaxis of human neutrophils. Key results: In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [125I]-interleukin-8 ([125I]-IL-8) without affecting the Kd. In contrast, IL-8 was unable to prevent binding of [3H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene α, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [35S]-GTPγS binding in this preparation. Conclusions and implications: Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling. PMID:19422399

Interaction of lafutidine in binding to human serum albumin in gastric ulcer therapy: STD-NMR, WaterLOGSY-NMR, NMR relaxation times, Tr-NOESY, molecule docking, and spectroscopic studies.

PubMed

Yang, Hongqin; Huang, Yanmei; He, Jiawei; Li, Shanshan; Tang, Bin; Li, Hui

2016-09-15

In this study, lafutidine (LAF) was used as a model compound to investigate the binding mechanism between antiulcer drugs and human serum albumin (HSA) through various techniques, including STD-NMR, WaterLOGSY-NMR, (1)H NMR relaxation times, tr-NOESY, molecule docking calculation, FT-IR spectroscopy, and CD spectroscopy. The analyses of STD-NMR, which derived relative STD (%) intensities, and WaterLOGSY-NMR, determined that LAF bound to HSA. In particular, the pyridyl group of LAF was in close contact with HSA binding pocket, whereas furyl group had a secondary binding. Competitive STD-NMR and WaterLOGSY-NMR experiments, with warifarin and ibuprofen as site-selective probes, indicated that LAF preferentially bound to site II in the hydrophobic subdomains IIIA of HSA. The bound conformation of LAF at the HSA binding site was further elucidated by transferred NOE effect (tr-NOESY) experiment. Relaxation experiments provided quantitative information about the relationship between the affinity and structure of LAF. The molecule docking simulations conducted with AutoDock and the restraints derived from STD results led to three-dimensional models that were consistent with the NMR spectroscopic data. The presence of hydrophobic forces and hydrogen interactions was also determined. Additionally, FT-IR and CD spectroscopies showed that LAF induced secondary structure changes of HSA. Copyright © 2016 Elsevier Inc. All rights reserved.

Allosteric binding sites in Rab11 for potential drug candidates

PubMed Central

2018-01-01

Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can preferentially bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may allosterically regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously. PMID:29874286

Kinetic analysis of central ( sup 11 C)raclopride binding to D2-dopamine receptors studied by PET--a comparison to the equilibrium analysis

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

Farde, L.; Eriksson, L.; Blomquist, G.

1989-10-01

(11C)Raclopride binding to central D2-dopamine receptors in humans has previously been examined by positron emission tomography (PET). Based on the rapid occurrence of binding equilibrium, a saturation analysis has been developed for the determination of receptor density (Bmax) and affinity (Kd). For analysis of PET measurements obtained with other ligands, a kinetic three-compartment model has been used. In the present study, the brain uptake of (11C)raclopride was analyzed further by applying both a kinetic and an equilibrium analysis to data obtained from four PET experiments in each of three healthy subjects. First regional CBV was determined. In the second andmore » third experiment, (11C)-raclopride with high and low specific activity was used. In a fourth experiment, the (11C)raclopride enantiomer (11C)FLB472 was used to examine the concentration of free radioligand and nonspecific binding in brain. Radio-activity in arterial blood was measured using an automated blood sampling system. Bmax and Kd values for (11C)raclopride binding could be determined also with the kinetic analysis. As expected theoretically, those values were similar to those obtained with the equilibrium analysis. In addition, the kinetic analysis allowed separate determination of the association and dissociation rate constants, kon and koff, respectively. Examination of (11C)raclopride and (11C)FLB472 uptake in brain regions devoid of specific D2-dopamine receptor binding indicated a fourth compartment in which uptake was reversible, nonstereoselective, and nonsaturable in the dose range studied.« less

Studies of the Binding of Modest Modulators of the Human Enzyme, Sirtuin 6, by STD NMR.

PubMed

Bolívar, Beatriz E; Welch, John T

2017-05-18

Pyrazinamide (PZA), an essential constituent of short-course tuberculosis chemotherapy, binds weakly but selectively to Sirtuin 6 (SIRT6). Despite the structural similarities between nicotinamide (NAM), PZA, and pyrazinoic acid (POA), these inhibitors modulate SIRT6 by different mechanisms and through different binding sites, as suggested by saturation transfer difference (STD) NMR. Available experimental evidence, such as that derived from crystal structures and kinetic experiments, has been of only limited utility in elucidation of the mechanistic details of sirtuin inhibition by NAM or other inhibitors. For instance, crystallographic structural analysis of sirtuin binding sites does not help us understand important differences in binding affinities among sirtuins or capture details of such dynamic process. Hence, STD NMR was utilized throughout this study. Our results not only agreed with the binding kinetics experiments but also gave a qualitative insight into the binding process. The data presented herein suggested some details about the geometry of the binding epitopes of the ligands in solution with the apo- and holoenzyme. Recognition that SIRT6 is affected selectively by PZA, an established clinical agent, suggests that the rational development of more potent and selective NAM surrogates might be possible. These derivatives might be accessible by employing the malleability of this scaffold to assist in the identification by STD NMR of the motifs that interact with the apo- and holoenzymes in solution. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3′′)(9) adenyltransferase

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

Chen, Yang; Näsvall, Joakim; Wu, Shiying

The crystal structure of the aminoglycoside-adenylating enzyme AadA is reported together with functional experiments providing insights into its oligomeric state, ligand binding and catalysis. Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundlemore » domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.« less

Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization.

PubMed

Ji, Xin; Olinger, Gene G; Aris, Sheena; Chen, Ying; Gewurz, Henry; Spear, Gregory T

2005-09-01

Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

Glycomic Characterization of Respiratory Tract Tissues of Ferrets

PubMed Central

Jia, Nan; Barclay, Wendy S.; Roberts, Kim; Yen, Hui-Ling; Chan, Renee W. Y.; Lam, Alfred K. Y.; Air, Gillian; Peiris, J. S. Malik; Dell, Anne; Nicholls, John M.; Haslam, Stuart M.

2014-01-01

The initial recognition between influenza virus and the host cell is mediated by interactions between the viral surface protein hemagglutinin and sialic acid-terminated glycoconjugates on the host cell surface. The sialic acid residues can be linked to the adjacent monosaccharide by α2–3- or α2–6-type glycosidic bonds. It is this linkage difference that primarily defines the species barrier of the influenza virus infection with α2–3 binding being associated with avian influenza viruses and α2–6 binding being associated with human strains. The ferret has been extensively used as an animal model to study the transmission of influenza. To better understand the validity of this model system, we undertook glycomic characterization of respiratory tissues of ferret, which allows a comparison of potential viral receptors to be made between humans and ferrets. To complement the structural analysis, lectin staining experiments were performed to characterize the regional distributions of glycans along the respiratory tract of ferrets. Finally, the binding between the glycans identified and the hemagglutinins of different strains of influenza viruses was assessed by glycan array experiments. Our data indicated that the respiratory tissues of ferret heterogeneously express both α2–3- and α2–6-linked sialic acids. However, the respiratory tissues of ferret also expressed the Sda epitope (NeuAcα2-3(GalNAcβ1–4)Galβ1–4GlcNAc) and sialylated N,N′-diacetyllactosamine (NeuAcα2–6GalNAcβ1–4GlcNAc), which have not been observed in the human respiratory tract surface epithelium. The presence of the Sda epitope reduces potential binding sites for avian viruses and thus may have implications for the usefulness of the ferret in the study of influenza virus infection. PMID:25135641

Glycomic characterization of respiratory tract tissues of ferrets: implications for its use in influenza virus infection studies.

PubMed

Jia, Nan; Barclay, Wendy S; Roberts, Kim; Yen, Hui-Ling; Chan, Renee W Y; Lam, Alfred K Y; Air, Gillian; Peiris, J S Malik; Dell, Anne; Nicholls, John M; Haslam, Stuart M

2014-10-10

The initial recognition between influenza virus and the host cell is mediated by interactions between the viral surface protein hemagglutinin and sialic acid-terminated glycoconjugates on the host cell surface. The sialic acid residues can be linked to the adjacent monosaccharide by α2-3- or α2-6-type glycosidic bonds. It is this linkage difference that primarily defines the species barrier of the influenza virus infection with α2-3 binding being associated with avian influenza viruses and α2-6 binding being associated with human strains. The ferret has been extensively used as an animal model to study the transmission of influenza. To better understand the validity of this model system, we undertook glycomic characterization of respiratory tissues of ferret, which allows a comparison of potential viral receptors to be made between humans and ferrets. To complement the structural analysis, lectin staining experiments were performed to characterize the regional distributions of glycans along the respiratory tract of ferrets. Finally, the binding between the glycans identified and the hemagglutinins of different strains of influenza viruses was assessed by glycan array experiments. Our data indicated that the respiratory tissues of ferret heterogeneously express both α2-3- and α2-6-linked sialic acids. However, the respiratory tissues of ferret also expressed the Sda epitope (NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAc) and sialylated N,N'-diacetyllactosamine (NeuAcα2-6GalNAcβ1-4GlcNAc), which have not been observed in the human respiratory tract surface epithelium. The presence of the Sda epitope reduces potential binding sites for avian viruses and thus may have implications for the usefulness of the ferret in the study of influenza virus infection. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Core Histones and HIRIP3, a Novel Histone-Binding Protein, Directly Interact with WD Repeat Protein HIRA

PubMed Central

Lorain, Stéphanie; Quivy, Jean-Pierre; Monier-Gavelle, Frédérique; Scamps, Christine; Lécluse, Yann; Almouzni, Geneviève; Lipinski, Marc

1998-01-01

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second α helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development. PMID:9710638