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

PubMed Central

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

2016-01-01

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

Smooth affine shear tight frames: digitization and applications

NASA Astrophysics Data System (ADS)

Zhuang, Xiaosheng

2015-08-01

In this paper, we mainly discuss one of the recent developed directional multiscale representation systems: smooth affine shear tight frames. A directional wavelet tight frame is generated by isotropic dilations and translations of directional wavelet generators, while an affine shear tight frame is generated by anisotropic dilations, shears, and translations of shearlet generators. These two tight frames are actually connected in the sense that the affine shear tight frame can be obtained from a directional wavelet tight frame through subsampling. Consequently, an affine shear tight frame indeed has an underlying filter bank from the MRA structure of its associated directional wavelet tight frame. We call such filter banks affine shear filter banks, which can be designed completely in the frequency domain. We discuss the digitization of affine shear filter banks and their implementations: the forward and backward digital affine shear transforms. Redundancy rate and computational complexity of digital affine shear transforms are also investigated in this paper. Numerical experiments and comparisons in image/video processing show the advantages of digital affine shear transforms over many other state-of-art directional multiscale representation systems.

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

PubMed Central

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

2015-01-01

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

The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

PubMed

Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

2012-09-01

Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

PubMed Central

Kaji, Tomohiro; Ishige, Akiko; Hikida, Masaki; Taka, Junko; Hijikata, Atsushi; Kubo, Masato; Nagashima, Takeshi; Takahashi, Yoshimasa; Kurosaki, Tomohiro; Okada, Mariko; Ohara, Osamu

2012-01-01

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell–dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell–dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen. PMID:23027924

High l-Trp affinity of indoleamine 2,3-dioxygenase 1 is attributed to two residues located in the distal heme pocket.

PubMed

Yuasa, Hajime J

2016-10-01

Indoleamine 2, 3-dioxygenase (IDO) catalyzes the oxidative cleavage of the pyrrole ring of l-Trp to generate N-formyl-kynurenine. Two IDO genes, IDO1 and IDO2, are found in vertebrates. Mammalian IDO1s are high-affinity, l-Trp-degrading enzymes, whereas IDO2s generally have a relatively low affinity. It has been suggested that the distal-Ser (corresponding to Ser167 of human IDO1) was crucial for improvement in the affinity for l-Trp but this idea was insufficient to explain the high affinity shown by mammalian IDO1. In this study, the amino acid sequences of vertebrate ancestral IDO1 and ancestral IDO2 were inferred, and bacterially expressed ancestral IDOs were characterized. Although the amino acid sequences of the enzymes shared high identity (86%) with each other, they showed distinct enzymatic properties. In analyses of a series of ancestral IDO1/IDO2 chimeric enzymes and their variants, the distal-Tyr (corresponding to Tyr126 of human IDO1) was detected as another and was probably the most crucial residue for high l-Trp affinity. The two amino acid substitutions (distal-Ser to Thr and distal-Tyr to His) drastically decreased the l-Trp affinity and catalytic efficiency of IDO1s. Conversely, two substitutions (distal-Thr to Ser and distal-His to Tyr) were sufficient to bestow IDO1-like high affinity on ancestral and chicken IDO2. © 2016 Federation of European Biochemical Societies.

High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

PubMed Central

2015-01-01

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.

PubMed

Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G

2014-04-04

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

Optically degradable dendrons for temporary adhesion of proteins to DNA.

PubMed

Kostiainen, Mauri A; Kotimaa, Juha; Laukkanen, Marja-Leena; Pavan, Giovanni M

2010-06-18

Experimental studies and molecular dynamics modeling demonstrate that multivalent dendrons can be used to temporarily glue proteins and DNA together with high affinity. We describe N-maleimide-cored polyamine dendrons that can be conjugated with free cysteine residues on protein surfaces through 1,4-conjugate addition to give one-to-one protein-polymer conjugates. We used a genetically engineered cysteine mutant of class II hydrophobin (HFBI) and a single-chain Fragment variable (scFv) antibody as model proteins for the conjugation reactions. The binding affinity of the protein-dendron conjugates towards DNA was experimentally assessed by using the ethidium bromide displacement assay. The binding was found to depend on the generation of the dendron, with the second generation having a stronger affinity than the first generation. Thermodynamic parameters of the binding were obtained from molecular dynamics modeling, which showed that the high binding affinity for each system is almost completely driven by a strong favorable binding enthalpy that is opposed by unfavorable binding entropy. A short exposure to UV (lambda approximately 350 nm) can cleave the photolabile o-nitrobenzyl-linked binding ligands from the surface of the dendron, which results in loss of the multivalent binding interactions and triggers the release of the DNA and protein. The timescale of the release is very rapid and the binding partners can be efficiently released after 3 min of UV exposure.

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

PubMed

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

2014-12-10

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. Copyright © 2014 Elsevier B.V. All rights reserved.

Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity

PubMed Central

Zandvakili, Arya; Campbell, Ian; Weirauch, Matthew T.

2018-01-01

Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs. PMID:29617378

Classical affine W-algebras associated to Lie superalgebras

NASA Astrophysics Data System (ADS)

Suh, Uhi Rinn

2016-02-01

In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalization of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.

Computational Design of Ligand Binding Proteins with High Affinity and Selectivity

PubMed Central

Dou, Jiayi; Doyle, Lindsey; Nelson, Jorgen W.; Schena, Alberto; Jankowski, Wojciech; Kalodimos, Charalampos G.; Johnsson, Kai; Stoddard, Barry L.; Baker, David

2014-01-01

The ability to design proteins with high affinity and selectivity for any given small molecule would have numerous applications in biosensing, diagnostics, and therapeutics, and is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition phenomena. Attempts to design ligand binding proteins have met with little success, however, and the computational design of precise molecular recognition between proteins and small molecules remains an “unsolved problem”1. We describe a general method for the computational design of small molecule binding sites with pre-organized hydrogen bonding and hydrophobic interfaces and high overall shape complementary to the ligand, and use it to design protein binding sites for the steroid digoxigenin (DIG). Of 17 designs that were experimentally characterized, two bind DIG; the highest affinity design has the lowest predicted interaction energy and the most pre-organized binding site in the set. A comprehensive binding-fitness landscape of this design generated by library selection and deep sequencing was used to guide optimization of binding affinity to a picomolar level, and two X-ray co-crystal structures of optimized complexes show atomic level agreement with the design models. The designed binder has a high selectivity for DIG over the related steroids digitoxigenin, progesterone, and β-estradiol, which can be reprogrammed through the designed hydrogen-bonding interactions. Taken together, the binding fitness landscape, co-crystal structures, and thermodynamic binding parameters illustrate how increases in binding affinity can result from distal sequence changes that limit the protein ensemble to conformers making the most energetically favorable interactions with the ligand. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics, and diagnostics. PMID:24005320

Classical affine W-algebras associated to Lie superalgebras

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

Suh, Uhi Rinn, E-mail: uhrisu1@math.snu.ac.kr

2016-02-15

In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalizationmore » of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.« less

Effect of Antipsychotic Type and Dose Changes on Tardive Dyskinesia and Parkinsonism Severity in Patients With a Serious Mental Illness: The Curaçao Extrapyramidal Syndromes Study XII.

PubMed

Mentzel, Charlotte L; Bakker, P Roberto; van Os, Jim; Drukker, Marjan; Matroos, Glenn E; Hoek, Hans W; Tijssen, Marina A J; van Harten, Peter N

2017-03-01

To test the efficacy of current treatment recommendations for parkinsonism and tardive dyskinesia (TD) severity in patients with severe mental illness (SMI). We present an 18-year prospective study including all 223 patients with SMI (as defined by the 1987 US National Institute of Mental Health, which were based on DSM-III-R diagnostic criteria) receiving care from the only psychiatric hospital of the former Netherlands Antilles. Eight clinical assessments (1992-2009) focused on movement disorders and medication use. Tardive dyskinesia was measured by the Abnormal Involuntary Movement Scale and parkinsonism by the Unified Parkinson's Disease Rating Scale. Antipsychotics were classified into first-generation antipsychotic (FGA) versus second-generation antipsychotic (SGA) and high versus low dopamine 2 (D₂) affinity categories. The effect that switching has within each category on subsequent movement scores was calculated separately by using time-lagged multilevel logistic regression models. There was a significant association between reduction in TD severity and starting/switching to an FGA (B = -3.54, P < .001) and starting/switching to a high D₂ affinity antipsychotic (B = -2.49, P < .01). Adding an SGA to existing FGA treatment was associated with reduction in TD severity (B = -2.43, P < .01). For parkinsonism, stopping antipsychotics predicted symptom reduction (B = -7.76, P < .01 in FGA/SGA-switch model; B = -7.74, P < .01 in D₂ affinity switch model), while starting a high D₂ affinity antipsychotic predicted an increase in symptoms (B = 3.29, P < .05 in D₂ affinity switch model). The results show that switching from an FGA to an SGA does not necessarily result in a reduction of TD or parkinsonism. Only stopping all antipsychotics reduces the severity of parkinsonism, and starting an FGA or a high D₂ affinity antipsychotic may reduce the severity of TD. © Copyright 2017 Physicians Postgraduate Press, Inc.

Concepts in receptor optimization: targeting the RGD peptide.

PubMed

Chen, Wei; Chang, Chia-en; Gilson, Michael K

2006-04-12

Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration* ♦

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F.; Fuh, Germaine

2015-01-01

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

Transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies.

PubMed

Green, Larry L

2014-03-01

Transgenic mice have yielded seven of the ten currently-approved human antibody drugs, making them the most successful platform for the discovery of fully human antibody therapeutics. The use of the in vivo immune system helps drive this success by taking advantage of the natural selection process that produces antibodies with desirable characteristics. Appropriately genetically-engineered mice act as robust engines for the generation of diverse repertoires of affinity- matured fully human variable regions with intrinsic properties necessary for successful antibody drug development including high potency, specificity, manufacturability, solubility and low risk of immunogenicity. A broad range of mAb drug targets are addressable in these mice, comprising both secreted and transmembrane targets, including membrane multi-spanning targets, as well as human target antigens that share high sequence identity with their mouse orthologue. Transgenic mice can routinely yield antibodies with sub-nanomolar binding affinity for their antigen, with lead candidate mAbs frequently possessing affinities for binding to their target of less than 100 picomolar, without requiring any ex vivo affinity optimization. While the originator transgenic mice platforms are no longer broadly available, a new generation of transgenic platforms is in development for discovery of the next wave of human therapeutic antibodies.

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen

PubMed Central

1995-01-01

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p- azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment. PMID:7650481

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

PubMed Central

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

Low-complexity piecewise-affine virtual sensors: theory and design

NASA Astrophysics Data System (ADS)

Rubagotti, Matteo; Poggi, Tomaso; Oliveri, Alberto; Pascucci, Carlo Alberto; Bemporad, Alberto; Storace, Marco

2014-03-01

This paper is focused on the theoretical development and the hardware implementation of low-complexity piecewise-affine direct virtual sensors for the estimation of unmeasured variables of interest of nonlinear systems. The direct virtual sensor is designed directly from measured inputs and outputs of the system and does not require a dynamical model. The proposed approach allows one to design estimators which mitigate the effect of the so-called 'curse of dimensionality' of simplicial piecewise-affine functions, and can be therefore applied to relatively high-order systems, enjoying convergence and optimality properties. An automatic toolchain is also presented to generate the VHDL code describing the digital circuit implementing the virtual sensor, starting from the set of measured input and output data. The proposed methodology is applied to generate an FPGA implementation of the virtual sensor for the estimation of vehicle lateral velocity, using a hardware-in-the-loop setting.

Higher spins and Yangian symmetries

DOE PAGES

Gaberdiel, Matthias R.; Gopakumar, Rajesh; Li, Wei; ...

2017-04-26

The relation between the bosonic higher spin W∞[λ]W∞[λ] algebra, the affine Yangian of gl 1, and the SH c algebra is established in detail. For generic λ we find explicit expressions for the low-lying W∞[λ] modes in terms of the affine Yangian generators, and deduce from this the precise identification between λ and the parameters of the affine Yangian. Furthermore, for the free field cases corresponding to λ = 0 and λ = 1 we give closed-form expressions for the affine Yangian generators in terms of the free fields. Interestingly, the relation between the W∞ modes and those of themore » affine Yangian is a non-local one, in general. We also establish the explicit dictionary between the affine Yangian and the SH c generators. Lastly, given that Yangian algebras are the hallmark of integrability, these identifications should pave the way towards uncovering the relation between the integrable and the higher spin symmetries.« less

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

Gaberdiel, Matthias R.; Gopakumar, Rajesh; Li, Wei

The relation between the bosonic higher spin W∞[λ]W∞[λ] algebra, the affine Yangian of gl 1, and the SH c algebra is established in detail. For generic λ we find explicit expressions for the low-lying W∞[λ] modes in terms of the affine Yangian generators, and deduce from this the precise identification between λ and the parameters of the affine Yangian. Furthermore, for the free field cases corresponding to λ = 0 and λ = 1 we give closed-form expressions for the affine Yangian generators in terms of the free fields. Interestingly, the relation between the W∞ modes and those of themore » affine Yangian is a non-local one, in general. We also establish the explicit dictionary between the affine Yangian and the SH c generators. Lastly, given that Yangian algebras are the hallmark of integrability, these identifications should pave the way towards uncovering the relation between the integrable and the higher spin symmetries.« less

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

PubMed

Pan, Yuchen; Sackmann, Eric K; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S; Herr, Amy E

2016-12-23

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (K D ) of each recombinant antibody and the target antigen. To characterize the K D of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The K D for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis

PubMed Central

Pan, Yuchen; Sackmann, Eric K.; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S.; Herr, Amy E.

2016-01-01

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality – the binding affinity – is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization. PMID:28008969

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

PubMed Central

Ballermann, B J; Hoover, R L; Karnovsky, M J; Brenner, B M

1985-01-01

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus. Images PMID:3001139

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

PubMed

Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

2015-09-04

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cellular Targets of Dietary Polyphenol Resveratrol

DTIC Science & Technology

2005-03-01

attempts to generate affinity columns tagged with other polyphenols, e.g., epigallocatechin gallate ( EGCG ). Conceivably such columns, if generated, would...Similar affinity chromatography with the related polyphenol Epigallocatechin gallate does not produce similar results.” Answer: We did not make...addition, the PI does not provid expression. If there is “increased ex many bind the resveratrol affinity co related polyphenol Epigallocatechin Response

High-affinity PD-1 molecules deliver improved interaction with PD-L1 and PD-L2.

PubMed

Li, Yanyan; Liang, Zhaoduan; Tian, Ye; Cai, Wenxuan; Weng, Zhiming; Chen, Lin; Zhang, Huanling; Bao, Yifeng; Zheng, Hongjun; Zeng, Sihai; Bei, Chunhua; Li, Yi

2018-06-11

The inhibitory checkpoint molecule programmed death (PD)-1 plays a vital role in maintaining immune homeostasis upon binding to its ligands, PD-L1 and PD-L2. Several recent studies have demonstrated that soluble PD-1 (sPD-1) can block the interaction between membrane PD-1 and PD-L1 to enhance the anti-tumor capability of T cells. However, the affinity of natural sPD-1 binding to PD-L1 is too low to permit therapeutic applications. Here a PD-1 variant with ~3,000-fold and ~70-fold affinity increase to bind PD-L1 and PD-L2, respectively, was generated through directed molecular evolution and phage display technology. Structural analysis showed that mutations at amino acid positions 124 and 132 of PD-1 played major roles in enhancing the affinity of PD-1 binding to its ligands. The high-affinity PD-1 mutant could compete with the binding of antibodies specific to PD-L1 or PD-L2 on cancer cells or dendritic cells (DCs), and it could enhance the proliferation and IFN-γ release of activated lymphocytes. These features potentially qualify the high-affinity PD-1 variant as a unique candidate for the development of a new class of PD-1 immune checkpoint blockade therapeutics. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

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

Fanning, Sean W.; Horn, James R.

2014-03-05

Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while themore » crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.« less

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

PubMed

Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

2017-05-13

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

A dynamic T cell–limited checkpoint regulates affinity-dependent B cell entry into the germinal center

PubMed Central

Schwickert, Tanja A.; Victora, Gabriel D.; Fooksman, David R.; Kamphorst, Alice O.; Mugnier, Monica R.; Gitlin, Alexander D.; Dustin, Michael L.

2011-01-01

The germinal center (GC) reaction is essential for the generation of the somatically hypermutated, high-affinity antibodies that mediate adaptive immunity. Entry into the GC is limited to a small number of B cell clones; however, the process by which this limited number of clones is selected is unclear. In this study, we demonstrate that low-affinity B cells intrinsically capable of seeding a GC reaction fail to expand and become activated in the presence of higher-affinity B cells even before GC coalescence. Live multiphoton imaging shows that selection is based on the amount of peptide–major histocompatibility complex (pMHC) presented to cognate T cells within clusters of responding B and T cells at the T–B border. We propose a model in which T cell help is restricted to the B cells with the highest amounts of pMHC, thus allowing for a dynamic affinity threshold to be imposed on antigen-binding B cells. PMID:21576382

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

Isolation of high-affinity, neutralizing anti-idiotype antibodies by phage and ribosome display for application in immunogenicity and pharmacokinetic analyses.

PubMed

Chin, Stacey E; Ferraro, Franco; Groves, Maria; Liang, Meina; Vaughan, Tristan J; Dobson, Claire L

2015-01-01

Anti-idiotype antibodies against a therapeutic antibody are key reagents for the development of immunogenicity and pharmacokinetic (PK) assays during pre-clinical and clinical development. Here we have used a combination of phage and ribosome display to isolate a panel of monoclonal anti-idiotype antibodies with sub-nanomolar affinity and high specificity to a human anti-IgE monoclonal antibody. Anti-idiotype antibodies were enriched from scFv libraries using phage display, and a biochemical epitope competition assay was used to identify anti-idiotypes which neutralized IgE binding, which was essential for the intended use of the anti-idiotypes as positive controls in neutralizing anti-drug antibody (Nab) assays. The phage display-derived anti-idiotype antibodies were rapidly affinity-matured using a random point mutagenesis approach in ribosome display. Ten anti-idiotype antibodies with improved neutralizing activity relative to the parent antibodies displayed sub-nanomolar affinity for the anti-IgE antibody, representing up to 20-fold improvements in affinity from just two rounds of affinity-based selection. The optimized anti-idiotype antibodies retained the specificity of the parent antibodies, and importantly, were fit for purpose for use in PK and anti-drug antibody (ADA) assays. The approach we describe here for generation of anti-idiotype antibodies to an anti-IgE antibody is generically applicable for the rapid isolation and affinity maturation of anti-idiotype antibodies to any antibody-based drug candidate. Copyright © 2014 Elsevier B.V. All rights reserved.

Flutriciclamide (18F-GE180) PET: First-in-Human PET Study of Novel Third-Generation In Vivo Marker of Human Translocator Protein.

PubMed

Fan, Zhen; Calsolaro, Valeria; Atkinson, Rebecca A; Femminella, Grazia D; Waldman, Adam; Buckley, Christopher; Trigg, William; Brooks, David J; Hinz, Rainer; Edison, Paul

2016-11-01

Neuroinflammation is associated with neurodegenerative disease. PET radioligands targeting the 18-kDa translocator protein (TSPO) have been used as in vivo markers of neuroinflammation, but there is an urgent need for novel probes with improved signal-to-noise ratio. Flutriciclamide ( 18 F-GE180) is a recently developed third-generation TSPO ligand. In this first study, we evaluated the optimum scan duration and kinetic modeling strategies for 18 F-GE180 PET in (older) healthy controls. Ten healthy controls, 6 TSPO high-affinity binders, and 4 mixed-affinity binders were recruited. All subjects underwent detailed neuropsychologic tests, MRI, and a 210-min 18 F-GE180 dynamic PET/CT scan using metabolite-corrected arterial plasma input function. We evaluated 5 different kinetic models: irreversible and reversible 2-tissue-compartment models, a reversible 1-tissue model, and 2 models with an extra irreversible vascular compartment. The minimal scan duration was established using 210-min scan data. The feasibility of generating parametric maps was also investigated using graphical analysis. 18 F-GE180 concentration was higher in plasma than in whole blood during the entire scan duration. The volume of distribution (V T ) was 0.17 in high-affinity binders and 0.12 in mixed-affinity binders using the kinetic model. The model that best represented brain 18 F-GE180 kinetics across regions was the reversible 2-tissue-compartment model (2TCM4k), and 90 min resulted as the optimum scan length required to obtain stable estimates. Logan graphical analysis with arterial input function gave a V T highly consistent with V T in the kinetic model, which could be used for voxelwise analysis. We report for the first time, to our knowledge, the kinetic properties of the novel third-generation TSPO PET ligand 18 F-GE180 in humans: 2TCM4k is the optimal method to quantify the brain uptake, 90 min is the optimal scan length, and the Logan approach could be used to generate parametric maps. Although these control subjects have shown relatively low V T , the methodology presented here forms the basis for quantification for future PET studies using 18 F-GE180 in different pathologies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Computational design of a pH-sensitive IgG binding protein.

PubMed

Strauch, Eva-Maria; Fleishman, Sarel J; Baker, David

2014-01-14

Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ∼ 4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

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

Hall, Justin; Brault, Amy; Vincent, Fabien

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a highmore » affinity (K D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.« less

Directed evolution of PDZ variants to generate high-affinity detection reagents.

PubMed

Ferrer, Marc; Maiolo, Jim; Kratz, Patricia; Jackowski, Jessica L; Murphy, Dennis J; Delagrave, Simon; Inglese, James

2005-04-01

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.

Inhibition of glycosylation on a camelid antibody uniquely affects its FcγRI binding activity.

PubMed

Krahn, Natalie; Spearman, Maureen; Meier, Markus; Dorion-Thibaudeau, July; McDougall, Matthew; Patel, Trushar R; De Crescenzo, Gregory; Durocher, Yves; Stetefeld, Jörg; Butler, Michael

2017-01-01

Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity are well known. However, little is understood about the effect that these modifications have on binding to the high affinity FcγRI receptor. This study analyzed the effect of variable N-glycosylation on a human-llama hybrid mAb (EG2-hFc, 80kDa) binding to FcγRI including a comparison to a full-sized IgG1 (DP-12, 150kDa). This was achieved by the addition of three glycosylation inhibitors (swainsonine, castanospermine, and kifunensine) independently to Chinese hamster ovary (CHO) cell cultures to generate hybrid and high mannose glycan structures. Biophysical analysis by circular dichroism, dynamic light scattering and analytical ultra-centrifugation confirmed that the solution-behaviour of the mAbs remained constant over multiple concentrations and glycan treatments. However, changes were observed when studying the interaction of FcγRI with variously glycosylated mAbs. Both mAbs were observed to have a decreased binding affinity upon treatment with swainsonine which produced hybrid glycans. Following de-glycosylation the binding affinity for EG2-hFc was only marginally reduced (6-fold) compared to a drastic (118-fold) decrease for DP-12. In summary, our data suggest that the relatively low molecular weight of chimeric EG2-hFc may contribute to its enhanced stability against glycan changes making it a highly suitable mAb candidate for therapeutic applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Urodilatin: binding properties and stimulation of cGMP generation in rat kidney cells.

PubMed

Saxenhofer, H; Fitzgibbon, W R; Paul, R V

1993-02-01

Urodilatin (URO) [ANP-(95-126)] is an analogue of atrial natriuretic peptide (alpha-ANP) [ANP-(99-126)] that was first isolated from human urine. In rat mesangial cells, URO competed with high affinity for non-guanylate cyclase-coupled ANPR-C receptors [concentration at which 50% labeled ligand is displaced (IC50) approximately 70 pM], but with lesser affinity to the guanylate cyclase-linked ANPR-A receptors (IC50 approximately 800 pM). alpha-ANP bound to both receptors with similar affinity [dissociation constant (Kd) approximately 150 pM]. In papillary collecting duct homogenates, which possess only ANPR-A receptors, the apparent Kd value averaged 229 pM for alpha-ANP and 2.7 nM for URO. Intravenous URO was at least as potent and effective as alpha-ANP in inducing diuresis and natriuresis in anesthetized rats, but URO was approximately 10-fold less potent in stimulating guanosine 3',5'-cyclic monophosphate generation in mesangial and inner medullary collecting duct cells. We conclude that URO has a lesser affinity than alpha-ANP for guanylate cyclase-coupled ANP receptors in the kidney and that the relative natriuretic potency of URO in vivo cannot be directly attributed to its binding characteristics with ANPR-A receptors.

PolyCheck: Dynamic Verification of Iteration Space Transformations on Affine Programs

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

Bao, Wenlei; Krishnamoorthy, Sriram; Pouchet, Louis-noel

2016-01-11

High-level compiler transformations, especially loop transformations, are widely recognized as critical optimizations to restructure programs to improve data locality and expose parallelism. Guaranteeing the correctness of program transformations is essential, and to date three main approaches have been developed: proof of equivalence of affine programs, matching the execution traces of programs, and checking bit-by-bit equivalence of the outputs of the programs. Each technique suffers from limitations in either the kind of transformations supported, space complexity, or the sensitivity to the testing dataset. In this paper, we take a novel approach addressing all three limitations to provide an automatic bug checkermore » to verify any iteration reordering transformations on affine programs, including non-affine transformations, with space consumption proportional to the original program data, and robust to arbitrary datasets of a given size. We achieve this by exploiting the structure of affine program control- and data-flow to generate at compile-time lightweight checker code to be executed within the transformed program. Experimental results assess the correctness and effectiveness of our method, and its increased coverage over previous approaches.« less

Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

PubMed Central

Rosa, Sofia Tabares-da; Rossotti, Martin; Carleiza, Carmen; Carrión, Federico; Pritsch, Otto; Ahn, Ki Chang; Last, Jerold A.; Hammock, Bruce D; González-Sapienza, Gualberto

2011-01-01

Single-domain antibodies (sdAbs) found in camelids, lack a light chain and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. While this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC50 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, KD 0.98–1.37 nM (SPR) which allowed development of a competitive assay for TCC with an IC50 = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC50 attained with other anti-hapten VHHs reported thus far. Despite the modest overall anti-hapten sdAbs response in llamas, a small subpopulation of high affinity VHHs are generated that can be isolated by carefully design of the selection process. PMID:21827167

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

PubMed

Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

2012-12-01

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

PubMed

Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

2012-10-12

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Method for resurrecting negative electron affinity photocathodes after exposure to an oxidizing gas

DOEpatents

Mulhollan, Gregory A; Bierman, John C

2012-10-30

A method by which negative electron affinity photocathodes (201), single crystal, amorphous, or otherwise ordered, can be made to recover their quantum yield following exposure to an oxidizing gas has been discovered. Conventional recovery methods employ the use of cesium as a positive acting agent (104). In the improved recovery method, an electron beam (205), sufficiently energetic to generate a secondary electron cloud (207), is applied to the photocathode in need of recovery. The energetic beam, through the high secondary electron yield of the negative electron affinity surface (203), creates sufficient numbers of low energy electrons which act on the reduced-yield surface so as to negate the effects of absorbed oxidizing atoms thereby recovering the quantum yield to a pre-decay value.

A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

PubMed Central

DeGrasse, Jeffrey A.

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide. PMID:22438927

A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B.

PubMed

DeGrasse, Jeffrey A

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

Notch and affinity boundaries in Drosophila.

PubMed

Herranz, Héctor; Milán, Marco

2006-02-01

Cells in multicellular organisms often do not intermingle freely with each other. Differential cell affinities can contribute to organizing cells into different tissues. Drosophila limbs and the vertebrate central nervous system are subdivided into compartments. Cells in adjacent compartments do not mix. Cell interactions mediated by Notch-family receptors have been implicated in the specification of these compartment boundaries. Two recent reports analyze the role of the Notch signaling pathway in the generation of an affinity boundary in the Drosophila wing. The first report analyzes the connection between Notch and the actin cytoskeleton. The second report analyzes the differential requirements of Notch and the transcription factor Suppressor of Hairless in generating the affinity boundary.

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

PubMed

Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph

2017-09-15

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

PubMed

Berry, L M; Adams, R; Airey, M; Bracher, M G; Bourne, T; Carrington, B; Cross, A S; Davies, G C G; Finney, H M; Foulkes, R; Gozzard, N; Griffin, R A; Hailu, H; Lamour, S D; Lawson, A D; Lightwood, D J; McKnight, A J; O'Dowd, V L; Oxbrow, A K F; Popplewell, A G; Shaw, S; Stephens, P E; Sweeney, B; Tomlinson, K L; Uhe, C; Palframan, R T

2009-02-01

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Nonlinear force propagation, anisotropic stiffening and non-affine relaxation in a model cytoskeleton

NASA Astrophysics Data System (ADS)

Mizuno, Daisuke; Head, David; Ikebe, Emi; Nakamasu, Akiko; Kinoshita, Suguru; Peijuan, Zhang; Ando, Shoji

2013-03-01

Forces are generated heterogeneously in living cells and transmitted through cytoskeletal networks that respond highly non-linearly. Here, we carry out high-bandwidth passive microrheology on vimentin networks reconstituted in vitro, and observe the nonlinear mechanical response due to forces propagating from a local source applied by an optical tweezer. Since the applied force is constant, the gel becomes equilibrated and the fluctuation-dissipation theorem can be employed to deduce the viscoelasticity of the local environment from the thermal fluctuations of colloidal probes. Our experiments unequivocally demonstrate the anisotropic stiffening of the cytoskeletal network behind the applied force, with greater stiffening in the parallel direction. Quantitative agreement with an affine continuum model is obtained, but only for the response at certain frequency ~ 10-1000 Hz which separates the high-frequency power law and low-frequency elastic behavior of the network. We argue that the failure of the model at lower frequencies is due to the presence of non-affinity, and observe that zero-frequency changes in particle separation can be fitted when an independently-measured, empirical nonaffinity factor is applied.

Cutting edge: double-stranded DNA breaks in the IgV region gene were detected at lower frequency in affinity-maturation impeded GANP-/- mice.

PubMed

Kawatani, Yousuke; Igarashi, Hideya; Matsui, Takeshi; Kuwahara, Kazuhiko; Fujimura, Satoru; Okamoto, Nobukazu; Takagi, Katsumasa; Sakaguchi, Nobuo

2005-11-01

Double-stranded DNA breaks (DSBs) at the IgV region (IgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken gamma-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP-/- B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANPTg) B cells showed an increased level. These results suggested that the level of IgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation.

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

PubMed

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

PubMed Central

Buenrostro, Jason D.; Chircus, Lauren M.; Araya, Carlos L.; Layton, Curtis J.; Chang, Howard Y.; Snyder, Michael P.; Greenleaf, William J.

2015-01-01

RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of MS2 coat protein to >107 RNA targets generated on a flow-cell surface by in situ transcription and inter-molecular tethering of RNA to DNA. We decompose the binding energy contributions from primary and secondary RNA structure, finding that differences in affinity are often driven by sequence-specific changes in association rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis, and a long-hypothesized structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNAMaP) relationships across molecular variants. PMID:24727714

Dual Targeting of the Chemokine Receptors CXCR4 and ACKR3 with Novel Engineered Chemokines*

PubMed Central

Hanes, Melinda S.; Salanga, Catherina L.; Chowdry, Arnab B.; Comerford, Iain; McColl, Shaun R.; Kufareva, Irina; Handel, Tracy M.

2015-01-01

The chemokine CXCL12 and its G protein-coupled receptors CXCR4 and ACKR3 are implicated in cancer and inflammatory and autoimmune disorders and are targets of numerous antagonist discovery efforts. Here, we describe a series of novel, high affinity CXCL12-based modulators of CXCR4 and ACKR3 generated by selection of N-terminal CXCL12 phage libraries on live cells expressing the receptors. Twelve of 13 characterized CXCL12 variants are full CXCR4 antagonists, and four have Kd values <5 nm. The new variants also showed high affinity for ACKR3. The variant with the highest affinity for CXCR4, LGGG-CXCL12, showed efficacy in a murine model for multiple sclerosis, demonstrating translational potential. Molecular modeling was used to elucidate the structural basis of binding and antagonism of selected variants and to guide future designs. Together, this work represents an important step toward the development of therapeutics targeting CXCR4 and ACKR3. PMID:26216880

Computational design of environmental sensors for the potent opioid fentanyl

DOE PAGES

Bick, Matthew J.; Greisen, Per J.; Morey, Kevin J.; ...

2017-09-19

Here, we describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We also use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

Computational design of environmental sensors for the potent opioid fentanyl

PubMed Central

Morey, Kevin J; Antunes, Mauricio S; La, David; Sankaran, Banumathi; Reymond, Luc; Johnsson, Kai; Medford, June I

2017-01-01

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment. PMID:28925919

Computational design of environmental sensors for the potent opioid fentanyl

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

Bick, Matthew J.; Greisen, Per J.; Morey, Kevin J.

Here, we describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We also use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

PubMed Central

Gabe, Claire M.; Brookes, Steven J.; Kirkham, Jennifer

2017-01-01

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond. PMID:28670287

Self-Assembly of Amphiphilic Dendrimers: The Role of Generation and Alkyl Chain Length in siRNA Interaction

PubMed Central

Márquez-Miranda, Valeria; Araya-Durán, Ingrid; Camarada, María Belén; Comer, Jeffrey; Valencia-Gallegos, Jesús A.; González-Nilo, Fernando Danilo

2016-01-01

An ideal nucleic-acid transfection system should combine the physical and chemical characteristics of cationic lipids and linear polymers to decrease cytotoxicity and uptake limitations. Previous research described new types of carriers termed amphiphilic dendrimers (ADs), which are based on polyamidoamine dendrimers (PAMAM). These ADs display the cell membrane affinity advantage of lipids and preserve the high affinity for DNA possessed by cationic dendrimers. These lipid/dendrimer hybrids consist of a low-generation, hydrophilic dendron (G2, G1, or G0) bonded to a hydrophobic tail. The G2-18C AD was reported to be an efficient siRNA vector with significant gene silencing. However, shorter tail ADs (G2-15C and G2-13C) and lower generation (G0 and G1) dendrimers failed as transfection carriers. To date, the self-assembly phenomenon of this class of amphiphilic dendrimers has not been molecularly explored using molecular simulation methods. To gain insight into these systems, the present study used coarse-grained molecular dynamics simulations to describe how ADs are able to self-assemble into an aggregate, and, specifically, how tail length and generation play a key role in this event. Finally, explanations are given for the better efficiency of G2/18-C as gene carrier in terms of binding of siRNA. This knowledge could be relevant for the design of novel, safer ADs with well-optimized affinity for siRNA. PMID:27377641

Fragment-based drug discovery using rational design.

PubMed

Jhoti, H

2007-01-01

Fragment-based drug discovery (FBDD) is established as an alternative approach to high-throughput screening for generating novel small molecule drug candidates. In FBDD, relatively small libraries of low molecular weight compounds (or fragments) are screened using sensitive biophysical techniques to detect their binding to the target protein. A lower absolute affinity of binding is expected from fragments, compared to much higher molecular weight hits detected by high-throughput screening, due to their reduced size and complexity. Through the use of iterative cycles of medicinal chemistry, ideally guided by three-dimensional structural data, it is often then relatively straightforward to optimize these weak binding fragment hits into potent and selective lead compounds. As with most other lead discovery methods there are two key components of FBDD; the detection technology and the compound library. In this review I outline the two main approaches used for detecting the binding of low affinity fragments and also some of the key principles that are used to generate a fragment library. In addition, I describe an example of how FBDD has led to the generation of a drug candidate that is now being tested in clinical trials for the treatment of cancer.

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

PubMed Central

Groves, Maria AT; Amanuel, Lily; Campbell, Jamie I; Rees, D Gareth; Sridharan, Sudharsan; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

2014-01-01

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics. PMID:24256948

01-ERD-111 - The Development of Synthetic High Affinity Ligands

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

Perkins, J; Balhorn, R; Cosman, M

2004-02-05

The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus ormore » botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.« less

Twin hydroxymethyluracil-A base pair steps define the binding site for the DNA-binding protein TF1.

PubMed

Grove, A; Figueiredo, M L; Galeone, A; Mayol, L; Geiduschek, E P

1997-05-16

The DNA-bending protein TF1 is the Bacillus subtilis bacteriophage SPO1-encoded homolog of the bacterial HU proteins and the Escherichia coli integration host factor. We recently proposed that TF1, which binds with high affinity (Kd was approximately 3 nM) to preferred sites within the hydroxymethyluracil (hmU)-containing phage genome, identifies its binding sites based on sequence-dependent DNA flexibility. Here, we show that two hmU-A base pair steps coinciding with two previously proposed sites of DNA distortion are critical for complex formation. The affinity of TF1 is reduced 10-fold when both of these hmU-A base pair steps are replaced with A-hmU, G-C, or C-G steps; only modest changes in affinity result when substitutions are made at other base pairs of the TF1 binding site. Replacement of all hmU residues with thymine decreases the affinity of TF1 greatly; remarkably, the high affinity is restored when the two hmU-A base pair steps corresponding to previously suggested sites of distortion are reintroduced into otherwise T-containing DNA. T-DNA constructs with 3-base bulges spaced apart by 9 base pairs of duplex also generate nM affinity of TF1. We suggest that twin hmU-A base pair steps located at the proposed sites of distortion are key to target site selection by TF1 and that recognition is based largely, if not entirely, on sequence-dependent DNA flexibility.

High-density platinum nanoparticle-decorated titanium dioxide nanofiber networks for efficient capillary photocatalytic hydrogen generation

Treesearch

Zhaodong Li; Chunhua Yao; Yi-Cheng Wang; Solomon Mikael; Sundaram Gunasekaran; Zhenqiang Ma; Zhiyong Cai; Xudong Wang

2016-01-01

Aldehyde-functionalized cellulose nanofibers (CNFs) were applied to synthesize Pt nanoparticles (NPs) on CNF surfaces via on-site Pt ion reduction and achieve high concentration and uniform Pt NP loading. ALD could then selectively deposit TiO2 on CNFs and keep the Pt NPs uncovered due to their drastically different hydro-affinity properties. The...

RAPID CLONING OF HIGH AFFINITY HUMAN MONOCLONAL ANTIBODIES AGAINST INFLUENZA VIRUS

PubMed Central

Wrammert, Jens; Smith, Kenneth; Miller, Joe; Langley, Trey; Kokko, Kenneth; Larsen, Christian; Zheng, Nai-Ying; Mays, Israel; Garman, Lori; Helms, Christina; James, Judith; Air, Gillian M.; Capra, J. Donald; Ahmed, Rafi; Wilson, Patrick C.

2008-01-01

Pre-existing neutralizing antibody provides the first line of defense against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14 to 21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B cell receptor (BCR) repertoire that in some donors were dominated by only a few B cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over fifty human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high affinity mAbs from humans within a month after vaccination. The panel of influenza virus specific human mAbs allowed us to address the issue of original antigenic sin (OAS) - the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared to the virus strain present in the vaccine1. However, we found that the vast majority of the influenza virus specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal healthy adults receiving influenza vaccination. PMID:18449194

Rapid cloning of high-affinity human monoclonal antibodies against influenza virus.

PubMed

Wrammert, Jens; Smith, Kenneth; Miller, Joe; Langley, William A; Kokko, Kenneth; Larsen, Christian; Zheng, Nai-Ying; Mays, Israel; Garman, Lori; Helms, Christina; James, Judith; Air, Gillian M; Capra, J Donald; Ahmed, Rafi; Wilson, Patrick C

2008-05-29

Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

PubMed

Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

2018-01-01

Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin

PubMed Central

Zhuang, Tiandi; Chen, Qiuyan; Cho, Min-Kyu; Vishnivetskiy, Sergey A.; Iverson, Tina M.; Gurevich, Vsevolod V.; Sanders, Charles R.

2013-01-01

Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (KD > 150 μM), its affinity for P-Rh (KD ∼80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (KD of ∼50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins. PMID:23277586

Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin.

PubMed

Zhuang, Tiandi; Chen, Qiuyan; Cho, Min-Kyu; Vishnivetskiy, Sergey A; Iverson, Tina M; Gurevich, Vsevolod V; Sanders, Charles R

2013-01-15

Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K(D) > 150 μM), its affinity for P-Rh (K(D) ~80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K(D) of~50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications.

PubMed

Kimoto, Michiko; Nakamura, Mana; Hirao, Ichiro

2016-09-06

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G-C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165 Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Synthesis of soybean oil-based thiol oligomers.

PubMed

Wu, Jennifer F; Fernando, Shashi; Weerasinghe, Dimuthu; Chen, Zhigang; Webster, Dean C

2011-08-22

Industrial grade soybean oil (SBO) and thiols were reacted to generate thiol-functionalized oligomers via a thermal, free radical initiated thiol-ene reaction between the SBO double bond moieties and the thiol functional groups. The effect of the reaction conditions, including thiol concentration, catalyst loading level, reaction time, and atmosphere, on the molecular weight and the conversion to the resultant soy-thiols were examined in a combinatorial high-throughput fashion using parallel synthesis, combinatorial FTIR, and rapid gel permeation chromatography (GPC). High thiol functionality and concentration, high thermal free radical catalyst concentration, long reaction time, and the use of a nitrogen reaction atmosphere were found to favor fast consumption of the SBO, and produced high molecular weight products. The thiol conversion during the reaction was inversely affected by a high thiol concentration, but was favored by a long reaction time and an air reaction atmosphere. These experimental observations were explained by the initial low affinity of the SBO and thiol, and the improved affinity between the generated soy-thiol oligomers and unreacted SBO during the reaction. The synthesized soy-thiol oligomers can be used for renewable thiol-ene UV curable materials and high molecular solids and thiourethane thermal cure materials. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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…

Concentration-Dependent Multiple Binding Sites on Saliva-Treated Hydroxyapatite for Streptococcus sanguis

PubMed Central

Gibbons, R. J.; Moreno, E. C.; Etherden, I.

1983-01-01

The influence of bacterial cell concentration on estimates of the number of binding sites and the affinity for the adsorption of a strain of Streptococcus sanguis to saliva-treated hydroxyapatite was determined, and the possible presence of multiple binding sites for this organism was tested. The range of concentrations of available bacteria varied from 4.7 × 106 to 5,960 × 106 cells per ml. The numbers of adsorbed bacteria increased over the entire range tested, but a suggestion of a break in an otherwise smooth adsorption isotherm was evident. Values for the number of binding sites and the affinity varied considerably depending upon the range of available bacterial concentrations used to estimate them; high correlation coefficients were obtained in all cases. The use of low bacterial cell concentrations yielded lower values for the number of sites and much higher values for the affinity constant than did the use of high bacterial cell concentrations. When data covering the entire range of bacterial concentrations were employed, values for the number of sites and the affinity were similar to those obtained by using only high bacterial cell concentrations. The simplest explanation for these results is that there are multiple binding sites for S. sanguis on saliva-treated hydroxyapatite surfaces. When present in low concentration, the streptococci evidently attach to more specific high-affinity sites which become saturated when higher bacterial concentrations are employed. The possibility of multiple binding sites was substantiated by comparing estimates of the adsorption parameters from a computer-simulated isotherm with those derived from the experimentally generated isotherm. A mathematical model describing bacterial adsorption to binary binding sites was further evidence for the existence of at least two classes of binding sites for S. sanguis. Far fewer streptococci adsorbed to experimental pellicles prepared from saliva depleted of bacterial aggregating activity when low numbers of streptococci were used, but the magnitude of this difference was considerably less when high streptococcal concentrations were employed. This suggests an association between salivary components which possess bacterial-aggregating activity and bacterial adsorption to high-affinity specific binding sites on saliva-treated hydroxyapatite surfaces. PMID:6822416

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage-display

PubMed Central

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K.

2012-01-01

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious and time consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13, the N-terminal Forkhead-associated domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1-strand was discovered to be essential for phage-display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermal stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20–25 mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage-display. PMID:22985966

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display.

PubMed

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K

2012-11-23

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious, and time-consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13 the N-terminal Forkhead-associated (FHA1) domain of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be nonfunctional due to misfolding in the bacterial periplasm. To overcome this limitation, we constructed a library of FHA1 variants by mutagenic PCR and isolated functional variants after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1 strand was discovered to be essential for phage display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermally stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20-25mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage display. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparative investigation of surface transfer doping of hydrogen terminated diamond by high electron affinity insulators

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

Verona, C.; Marinelli, Marco; Verona-Rinati, G.

We report on a comparative study of transfer doping of hydrogenated single crystal diamond surface by insulators featured by high electron affinity, such as Nb{sub 2}O{sub 5}, WO{sub 3}, V{sub 2}O{sub 5}, and MoO{sub 3}. The low electron affinity Al{sub 2}O{sub 3} was also investigated for comparison. Hole transport properties were evaluated in the passivated hydrogenated diamond films by Hall effect measurements, and were compared to un-passivated diamond films (air-induced doping). A drastic improvement was observed in passivated samples in terms of conductivity, stability with time, and resistance to high temperatures. The efficiency of the investigated insulators, as electron acceptingmore » materials in hydrogenated diamond surface, is consistent with their electronic structure. These surface acceptor materials generate a higher hole sheet concentration, up to 6.5 × 10{sup 13} cm{sup −2}, and a lower sheet resistance, down to 2.6 kΩ/sq, in comparison to the atmosphere-induced values of about 1 × 10{sup 13} cm{sup −2} and 10 kΩ/sq, respectively. On the other hand, hole mobilities were reduced by using high electron affinity insulator dopants. Hole mobility as a function of hole concentration in a hydrogenated diamond layer was also investigated, showing a well-defined monotonically decreasing trend.« less

Accuracy Analysis on Large Blocks of High Resolution Images

NASA Technical Reports Server (NTRS)

Passini, Richardo M.

2007-01-01

Although high altitude frequencies effects are removed at the time of basic image generation, low altitude (Yaw) effects are still present in form of affinity/angular affinity. They are effectively removed by additional parameters. Bundle block adjustment based on properly weighted ephemeris/altitude quaternions (BBABEQ) are not enough to remove the systematic effect. Moreover, due to the narrow FOV of the HRSI, position and altitude are highly correlated making it almost impossible to separate and remove their systematic effects without extending the geometric model (Self-Calib.) The systematic effects gets evident on the increase of accuracy (in terms of RMSE at GCPs) for looser and relaxed ground control at the expense of large and strong block deformation with large residuals at check points. Systematic errors are most freely distributed and their effects propagated all over the block.

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

PubMed

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

2018-05-30

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

Converting One-Face α-Helix Mimetics into Amphiphilic α-Helix Mimetics as Potent Inhibitors of Protein-Protein Interactions.

PubMed

Lee, Ji Hoon; Oh, Misook; Kim, Hyun Soo; Lee, Huisun; Im, Wonpil; Lim, Hyun-Suk

2016-01-11

Many biologically active α-helical peptides adopt amphiphilic helical structures that contain hydrophobic residues on one side and hydrophilic residues on the other side. Therefore, α-helix mimetics capable of mimicking such amphiphilic helical peptides should possess higher binding affinity and specificity to target proteins. Here we describe an efficient method for generating amphiphilic α-helix mimetics. One-face α-helix mimetics having hydrophobic side chains on one side was readily converted into amphiphilic α-helix mimetics by introducing appropriate charged residues on the opposite side. We also demonstrate that such two-face amphiphilic α-helix mimetics indeed show remarkably improved binding affinity to a target protein, compared to one-face hydrophobic α-helix mimetics. We believe that generating a large combinatorial library of these amphiphilic α-helix mimetics can be valuable for rapid discovery of highly potent and specific modulators of protein-protein interactions.

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

Cancer.gov

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

Characterization of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.

PubMed

Talkad, V D; Patto, R J; Metz, D C; Turner, R J; Fortune, K P; Bhat, S T; Gardner, J D

1994-10-20

By measuring binding of [125I]CCK-8 and [3H]L-364,718 to rat pancreatic acini we demonstrated directly that the pancreatic CCK receptor can exist in three different affinity states with respect to CCK--high affinity, low affinity and very low affinity. Binding of [125I]CCK-8 reflects interaction of the tracer with the high and low affinity states, whereas binding of [3H]L-364,718 reflects interaction of the tracer with the low and very low affinity states. Treating acini with carbachol abolished the high affinity state of the CCK receptor and converted approximately 25% of the low affinity receptors to the very low affinity state. Carbachol treatment was particularly useful in establishing the values of Kd for the high and low affinity states for different CCK receptor agonists and antagonists. Of the various CCK receptor agonists tested, CCK-8 had the highest affinity for the high affinity state (Kd approximately 1 nM), whereas CCK-JMV-180 had the highest affinity for the low (Kd 7 nM) and very low affinity (Kd 200 nM) states. Gastrin and de(SO4)CCK-8 had affinities for the high and low affinity states of the receptor that were 100- to 400-fold less than those of CCK-8 but had affinities for the very low affinity state that were only 3- to 10-fold less than that of CCK-8. CCK receptor antagonists showed several patterns in interacting with the different states of the CCK receptor. L-364,718 had the same affinity for each state of the CCK receptor. CR1409 and Bt2cGMP each had similar affinities for the high and low affinity states and lower affinity for the very low affinity state. L-365,260 and CCK-JMV-179 had the highest affinity for the low affinity state and lower affinities for the high and very low affinity states. Different CCK receptor agonists caused the same maximal stimulation of amylase secretion but showed different degrees of amplification in terms of the relationship between their abilities to stimulate amylase secretion and their abilities to occupy the low affinity state of the CCK receptor. When amplification was expressed quantitatively as the value of Kd for the low affinity state divided by the corresponding EC50 for stimulating amylase secretion the values were CCK-8 (1000), de(SO)CCK-8 (1500), gastrin (100) and CCK-JMV-180 (Menozzi, D., Vinayek, R., Jensen, R.T. and Gardner, J.D. (1991) J. Biol. Chem. 266, 10385-1091).(ABSTRACT TRUNCATED AT 400 WORDS)

Brain tumor detection and segmentation in a CRF (conditional random fields) framework with pixel-pairwise affinity and superpixel-level features.

PubMed

Wu, Wei; Chen, Albert Y C; Zhao, Liang; Corso, Jason J

2014-03-01

Detection and segmentation of a brain tumor such as glioblastoma multiforme (GBM) in magnetic resonance (MR) images are often challenging due to its intrinsically heterogeneous signal characteristics. A robust segmentation method for brain tumor MRI scans was developed and tested. Simple thresholds and statistical methods are unable to adequately segment the various elements of the GBM, such as local contrast enhancement, necrosis, and edema. Most voxel-based methods cannot achieve satisfactory results in larger data sets, and the methods based on generative or discriminative models have intrinsic limitations during application, such as small sample set learning and transfer. A new method was developed to overcome these challenges. Multimodal MR images are segmented into superpixels using algorithms to alleviate the sampling issue and to improve the sample representativeness. Next, features were extracted from the superpixels using multi-level Gabor wavelet filters. Based on the features, a support vector machine (SVM) model and an affinity metric model for tumors were trained to overcome the limitations of previous generative models. Based on the output of the SVM and spatial affinity models, conditional random fields theory was applied to segment the tumor in a maximum a posteriori fashion given the smoothness prior defined by our affinity model. Finally, labeling noise was removed using "structural knowledge" such as the symmetrical and continuous characteristics of the tumor in spatial domain. The system was evaluated with 20 GBM cases and the BraTS challenge data set. Dice coefficients were computed, and the results were highly consistent with those reported by Zikic et al. (MICCAI 2012, Lecture notes in computer science. vol 7512, pp 369-376, 2012). A brain tumor segmentation method using model-aware affinity demonstrates comparable performance with other state-of-the art algorithms.

Evaluation of first generation synthetic cannabinoids on binding at non-cannabinoid receptors and in a battery of in vivo assays in mice

PubMed Central

Wiley, Jenny L.; Lefever, Timothy W.; Marusich, Julie A.; Grabenauer, Megan; Moore, Katherine N.; Huffman, John W.; Thomas, Brian F.

2016-01-01

Anecdotal reports suggest that abused synthetic cannabinoids produce cannabis-like “highs,” but some of their effects may also differ from traditional cannabinoids such as Δ9-tetrahydrocannabinol (THC). This study examined the binding affinities of first-generation indole-derived synthetic cannabinoids at cannabinoid and noncannabinoid receptors and their effects in a functional observational battery (FOB) and drug discrimination in mice. All seven compounds, except JWH-391, had favorable affinity (≤ 159 nM) for both cannabinoid receptors. In contrast, binding at noncannabinoid receptors was absent or weak. In the FOB, THC and the six active compounds disrupted behaviors in CNS activation and muscle tone/equilibrium domains. Unlike THC, however, synthetic cannabinoids impaired behavior across a wider dose and domain range, producing autonomic effects and signs of CNS excitability and sensorimotor reactivity. In addition, mice acquired JWH-018 discrimination, and THC and JWH-073 produced full substitution whereas the 5-HT2B antagonist mianserin did not substitute in mice trained to discriminate JWH-018 or THC. Urinary metabolite analysis showed that the compounds were extensively metabolized, with metabolites that could contribute to their in vivo effects. Together, these results show that, while first-generation synthetic cannabinoids shared some effects that were similar to those of THC, they also possessed effects that differed from traditional cannabinoids. The high nanomolar (or absent) affinities of these compounds at receptors for most major neurotransmitters suggests that these divergent effects may be related to the greater potencies and/or efficacies at CB1 receptors; however, action(s) at noncannabinoid receptors yet to be assessed or via different signaling pathways cannot be ruled out. PMID:27449567

Evaluation of first generation synthetic cannabinoids on binding at non-cannabinoid receptors and in a battery of in vivo assays in mice.

PubMed

Wiley, Jenny L; Lefever, Timothy W; Marusich, Julie A; Grabenauer, Megan; Moore, Katherine N; Huffman, John W; Thomas, Brian F

2016-11-01

Anecdotal reports suggest that abused synthetic cannabinoids produce cannabis-like "highs," but some of their effects may also differ from traditional cannabinoids such as Δ(9)-tetrahydrocannabinol (THC). This study examined the binding affinities of first-generation indole-derived synthetic cannabinoids at cannabinoid and noncannabinoid receptors and their effects in a functional observational battery (FOB) and drug discrimination in mice. All seven compounds, except JWH-391, had favorable affinity (≤159 nM) for both cannabinoid receptors. In contrast, binding at noncannabinoid receptors was absent or weak. In the FOB, THC and the six active compounds disrupted behaviors in CNS activation and muscle tone/equilibrium domains. Unlike THC, however, synthetic cannabinoids impaired behavior across a wider dose and domain range, producing autonomic effects and signs of CNS excitability and sensorimotor reactivity. In addition, mice acquired JWH-018 discrimination, and THC and JWH-073 produced full substitution whereas the 5-HT2B antagonist mianserin did not substitute in mice trained to discriminate JWH-018 or THC. Urinary metabolite analysis showed that the compounds were extensively metabolized, with metabolites that could contribute to their in vivo effects. Together, these results show that, while first-generation synthetic cannabinoids shared some effects that were similar to those of THC, they also possessed effects that differed from traditional cannabinoids. The high nanomolar (or absent) affinities of these compounds at receptors for most major neurotransmitters suggests that these divergent effects may be related to the greater potencies and/or efficacies at CB1 receptors; however, action(s) at noncannabinoid receptors yet to be assessed or via different signaling pathways cannot be ruled out. Copyright © 2016 Elsevier Ltd. All rights reserved.

Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library.

PubMed

Shahi, B; Mousavi Gargari, S L; Rasooli, I; Rajabi Bazl, M; Hoseinpoor, R

2014-08-01

To construct secondary mutant phage-display library of recombinant single variable domain (VHH) against botulinum neurotoxin E by error-prone PCR. The gene coding for specific VHH derived from the camel immunized with binding domain of botulinum neurotoxin E (BoNT/E) was amplified by error-prone PCR. Several biopanning rounds were used to screen the phage-displaying BoNT/E Hc nanobodies. The final nanobody, SHMR4, with increased affinity recognized BoNT/E toxin with no cross-reactivity with other antigens especially with related BoNT toxins. The constructed nanobody could be a suitable candidate for VHH-based biosensor production to detect the Clostridium botulinum type E. Diagnosis and treatment of botulinum neurotoxins are important. Generation of high-affinity antibodies based on the construction of secondary libraries using affinity maturation step leads to the development of reagents for precise diagnosis and therapy. © 2014 The Society for Applied Microbiology.

Quantitative structure-activity relationships of selective antagonists of glucagon receptor using QuaSAR descriptors.

PubMed

Manoj Kumar, Palanivelu; Karthikeyan, Chandrabose; Hari Narayana Moorthy, Narayana Subbiah; Trivedi, Piyush

2006-11-01

In the present paper, quantitative structure activity relationship (QSAR) approach was applied to understand the affinity and selectivity of a novel series of triaryl imidazole derivatives towards glucagon receptor. Statistically significant and highly predictive QSARs were derived for glucagon receptor inhibition by triaryl imidazoles using QuaSAR descriptors of molecular operating environment (MOE) employing computer-assisted multiple regression procedure. The generated QSAR models revealed that factors related to hydrophobicity, molecular shape and geometry predominantly influences glucagon receptor binding affinity of the triaryl imidazoles indicating the relevance of shape specific steric interactions between the molecule and the receptor. Further, QSAR models formulated for selective inhibition of glucagon receptor over p38 mitogen activated protein (MAP) kinase of the compounds in the series highlights that the same structural features, which influence the glucagon receptor affinity, also contribute to their selective inhibition.

Rugged spin-polarized electron sources based on negative electron affinity GaAs photocathode with robust Cs2Te coating

NASA Astrophysics Data System (ADS)

Bae, Jai Kwan; Cultrera, Luca; DiGiacomo, Philip; Bazarov, Ivan

2018-04-01

Photocathodes capable of providing high intensity and highly spin-polarized electron beams with long operational lifetimes are of great interest for the next generation nuclear physics facilities like Electron Ion Colliders. We report on GaAs photocathodes activated by Cs2Te, a material well known for its robustness. GaAs activated by Cs2Te forms Negative Electron Affinity, and the lifetime for extracted charge is improved by a factor of 5 compared to that of GaAs activated by Cs and O2. The spin polarization of photoelectrons was measured using a Mott polarimeter and found to be independent from the activation method, thereby shifting the paradigm on spin-polarized electron sources employing photocathodes with robust coatings.

Importance of target-mediated drug disposition for small molecules.

PubMed

Smith, Dennis A; van Waterschoot, Robert A B; Parrott, Neil J; Olivares-Morales, Andrés; Lavé, Thierry; Rowland, Malcolm

2018-06-18

Target concentration is typically not considered in drug discovery. However, if targets are expressed at relatively high concentrations and compounds have high affinity, such that most of the drug is bound to its target, in vitro screens can give unreliable information on compound affinity. In vivo, a similar situation will generate pharmacokinetic (PK) profiles that deviate greatly from those normally expected, owing to target binding affecting drug distribution and clearance. Such target-mediated drug disposition (TMDD) effects on small molecules have received little attention and might only become apparent during clinical trials, with the potential for data misinterpretation. TMDD also confounds human microdosing approaches by providing therapeutically unrepresentative PK profiles. Being aware of these phenomena will improve the likelihood of successful drug discovery and development. Copyright © 2018. Published by Elsevier Ltd.

Detection of biomarkers using recombinant antibodies coupled to nanostructured platforms

PubMed Central

Kierny, Michael R.; Cunningham, Thomas D.; Kay, Brian K.

2012-01-01

The utility of biomarker detection in tomorrow's personalized health care field will mean early and accurate diagnosis of many types of human physiological conditions and diseases. In the search for biomarkers, recombinant affinity reagents can be generated to candidate proteins or post-translational modifications that differ qualitatively or quantitatively between normal and diseased tissues. The use of display technologies, such as phage-display, allows for manageable selection and optimization of affinity reagents for use in biomarker detection. Here we review the use of recombinant antibody fragments, such as scFvs and Fabs, which can be affinity-selected from phage-display libraries, to bind with both high specificity and affinity to biomarkers of cancer, such as Human Epidermal growth factor Receptor 2 (HER2) and Carcinoembryonic antigen (CEA). We discuss how these recombinant antibodies can be fabricated into nanostructures, such as carbon nanotubes, nanowires, and quantum dots, for the purpose of enhancing detection of biomarkers at low concentrations (pg/mL) within complex mixtures such as serum or tissue extracts. Other sensing technologies, which take advantage of ‘Surface Enhanced Raman Scattering’ (gold nanoshells), frequency changes in piezoelectric crystals (quartz crystal microbalance), or electrical current generation and sensing during electrochemical reactions (electrochemical detection), can effectively provide multiplexed platforms for detection of cancer and injury biomarkers. Such devices may soon replace the traditional time consuming ELISAs and Western blots, and deliver rapid, point-of-care diagnostics to market. PMID:22833780

Deconvolution of antibody affinities and concentrations by non-linear regression analysis of competitive ELISA data.

PubMed

Stevens, F J; Bobrovnik, S A

2007-12-01

Physiological responses of the adaptive immune system are polyclonal in nature whether induced by a naturally occurring infection, by vaccination to prevent infection or, in the case of animals, by challenge with antigen to generate reagents of research or commercial significance. The composition of the polyclonal responses is distinct to each individual or animal and changes over time. Differences exist in the affinities of the constituents and their relative proportion of the responsive population. In addition, some of the antibodies bind to different sites on the antigen, whereas other pairs of antibodies are sterically restricted from concurrent interaction with the antigen. Even if generation of a monoclonal antibody is the ultimate goal of a project, the quality of the resulting reagent is ultimately related to the characteristics of the initial immune response. It is probably impossible to quantitatively parse the composition of a polyclonal response to antigen. However, molecular regression allows further parameterization of a polyclonal antiserum in the context of certain simplifying assumptions. The antiserum is described as consisting of two competing populations of high- and low-affinity and unknown relative proportions. This simple model allows the quantitative determination of representative affinities and proportions. These parameters may be of use in evaluating responses to vaccines, to evaluating continuity of antibody production whether in vaccine recipients or animals used for the production of antisera, or in optimizing selection of donors for the production of monoclonal antibodies.

Recombinant human antibody fragment against tetanus toxoid produced by phage display.

PubMed

Neelakantam, B; Sridevi, N V; Shukra, A M; Sugumar, P; Samuel, S; Rajendra, L

2014-03-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen.

Enhanced ADCC Activity of Affinity Maturated and Fc-Engineered Mini-Antibodies Directed against the AML Stem Cell Antigen CD96

PubMed Central

Kellner, Christian; Bräutigam, Joachim; Staudinger, Matthias; Schub, Natalie; Peipp, Matthias; Gramatzki, Martin; Humpe, Andreas

2012-01-01

CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential. PMID:22879978

Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats.

PubMed

Pershad, Kritika; Sullivan, Mark A; Kay, Brian K

2011-05-15

Affinity reagents that are generated by phage display are typically subcloned into an expression vector for further biochemical characterization. This insert transfer process is time consuming and laborious especially if many inserts are to be subcloned. To simplify the transfer process, we have constructed a "drop-out" phagemid vector that can be rapidly converted to an expression vector by a simple restriction enzyme digestion with MfeI (to "drop-out" the gene III coding sequence), which generates alkaline phosphatase (AP) fusions of the affinity reagents on religation. Subsequently, restriction digestion with AscI drops out the AP coding region and religation generates affinity reagents with a C-terminal six-histidine tag. To validate the usefulness of this vector, four different human single chain Fragments of variable regions (scFv) were tested, three of which show specific binding to three zebrafish (Danio rerio) proteins, namely suppression of tumorigenicity 13, recoverin, and Ppib and the fourth binds to human Lactoferrin protein. For each of the constructs tested, the gene III and AP drop-out efficiency was between 90% and 100%. This vector is especially useful in speeding up the downstream screening of affinity reagents and bypassing the time-consuming subcloning experiments. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of Useful Nanobodies by Phage Display of Immune Single Domain Libraries Derived from Camelid Heavy Chain Antibodies.

PubMed

Romao, Ema; Morales-Yanez, Francisco; Hu, Yaozhong; Crauwels, Maxine; De Pauw, Pieter; Hassanzadeh, Gholamreza Ghassanzadeh; Devoogdt, Nick; Ackaert, Chloe; Vincke, Cecile; Muyldermans, Serge

2016-01-01

The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding fragments, also known as Nanobodies. Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display, bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify Nanobodies. The antigen affinity, stability, expression yields and structural details of the Nanobodies have been determined by standard technology. Nanobodies were subsequently engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin domains, or to add designed effector functions. Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm. The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen surface. Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been developed in the past decade. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Design, synthesis, and biological characterization of metabolically stable selective androgen receptor modulators.

PubMed

Marhefka, Craig A; Gao, Wenqing; Chung, Kiwon; Kim, Juhyun; He, Yali; Yin, Donghua; Bohl, Casey; Dalton, James T; Miller, Duane D

2004-02-12

A series of nonsteroidal ligands were synthesized as second-generation agonists for the androgen receptor (AR). These ligands were designed to eliminate metabolic sites identified in one of our first-generation AR agonists, which was inactive in vivo due to its rapid metabolism to inactive constituents. The binding affinity of these compounds was evaluated using AR isolated from rat ventral prostate. These second-generation compounds bound the AR in a high affinity and stereoselective manner, with K(i) values ranging from about 4 to 130 nM. The ability of these ligands to stimulate AR-mediated transcriptional activation was examined in cells transfected with the human AR and a hormone-dependent luciferase reporter gene. Although some compounds were unable to stimulate AR-mediated transcription, several demonstrated activity similar to that of dihydrotestosterone (DHT, an endogenous steroidal ligand for the AR). We also evaluated the in vivo pharmacologic activity of selected compounds in castrated male rats. Three compounds were identified as selective androgen receptor modulators (SARMs), exhibiting significant anabolic activity while having only moderate to minimal androgenic activity in vivo.

Design, Synthesis, and Biological Characterization of Metabolically Stable Selective Androgen Receptor Modulators

PubMed Central

Marhefka, Craig A.; Gao, Wenqing; Chung, Kiwon; Kim, Juhyun; He, Yali; Yin, Donghua; Bohl, Casey; Dalton, James T.; Miller, Duane D.

2007-01-01

A series of nonsteroidal ligands were synthesized as second-generation agonists for the androgen receptor (AR). These ligands were designed to eliminate metabolic sites identified in one of our first-generation AR agonists, which was inactive in vivo due to its rapid metabolism to inactive constituents. The binding affinity of these compounds was evaluated using AR isolated from rat ventral prostate. These second-generation compounds bound the AR in a high affinity and stereoselective manner, with Ki values ranging from about 4 to 130 nM. The ability of these ligands to stimulate AR-mediated transcriptional activation was examined in cells transfected with the human AR and a hormone-dependent luciferase reporter gene. Although some compounds were unable to stimulate AR-mediated transcription, several demonstrated activity similar to that of dihydrotestosterone (DHT, an endogenous steroidal ligand for the AR). We also evaluated the in vivo pharmacologic activity of selected compounds in castrated male rats. Three compounds were identified as selective androgen receptor modulators (SARMs), exhibiting significant anabolic activity while having only moderate to minimal androgenic activity in vivo. PMID:14761201

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

Suh, Uhi Rinn, E-mail: uhrisu1@math.snu.ac.kr

We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms ofmore » free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.« less

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

PubMed Central

Thie, Holger

2017-01-01

ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

Affine cellularity of finite type KLR algebras, and homomorphisms between Specht modules for KLR algebras in affine type A

NASA Astrophysics Data System (ADS)

Loubert, Joseph William

This thesis consists of two parts. In the first we prove that the Khovanov-Lauda-Rouquier algebras Ralpha of finite type are (graded) affine cellular in the sense of Koenig and Xi. In fact, we establish a stronger property, namely that the affine cell ideals in Ralpha are generated by idempotents. This in particular implies the (known) result that the global dimension of Ralpha is finite. In the second part we use the presentation of the Specht modules given by Kleshchev-Mathas-Ram to derive results about Specht modules. In particular, we determine all homomorphisms from an arbitrary Specht module to a fixed Specht module corresponding to any hook partition. Along the way, we give a complete description of the action of the standard KLR generators on the hook Specht module. This work generalizes a result of James. This dissertation includes previously published coauthored material.

AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

PubMed

Holland, Erika G; Buhr, Diane L; Acca, Felicity E; Alderman, Dawn; Bovat, Kristin; Busygina, Valeria; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

2013-08-30

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes. Copyright © 2013. Published by Elsevier B.V.

Advancements in Aptamer Discovery Technologies.

PubMed

Gotrik, Michael R; Feagin, Trevor A; Csordas, Andrew T; Nakamoto, Margaret A; Soh, H Tom

2016-09-20

Affinity reagents that specifically bind to their target molecules are invaluable tools in nearly every field of modern biomedicine. Nucleic acid-based aptamers offer many advantages in this domain, because they are chemically synthesized, stable, and economical. Despite these compelling features, aptamers are currently not widely used in comparison to antibodies. This is primarily because conventional aptamer-discovery techniques such as SELEX are time-consuming and labor-intensive and often fail to produce aptamers with comparable binding performance to antibodies. This Account describes a body of work from our laboratory in developing advanced methods for consistently producing high-performance aptamers with higher efficiency, fewer resources, and, most importantly, a greater probability of success. We describe our efforts in systematically transforming each major step of the aptamer discovery process: selection, analysis, and characterization. To improve selection, we have developed microfluidic devices (M-SELEX) that enable discovery of high-affinity aptamers after a minimal number of selection rounds by precisely controlling the target concentration and washing stringency. In terms of improving aptamer pool analysis, our group was the first to use high-throughput sequencing (HTS) for the discovery of new aptamers. We showed that tracking the enrichment trajectory of individual aptamer sequences enables the identification of high-performing aptamers without requiring full convergence of the selected aptamer pool. HTS is now widely used for aptamer discovery, and open-source software has become available to facilitate analysis. To improve binding characterization, we used HTS data to design custom aptamer arrays to measure the affinity and specificity of up to ∼10(4) DNA aptamers in parallel as a means to rapidly discover high-quality aptamers. Most recently, our efforts have culminated in the invention of the "particle display" (PD) screening system, which transforms solution-phase aptamers into "aptamer particles" that can be individually screened at high-throughput via fluorescence-activated cell sorting. Using PD, we have shown the feasibility of rapidly generating aptamers with exceptional affinities, even for proteins that have previously proven intractable to aptamer discovery. We are confident that these advanced aptamer-discovery methods will accelerate the discovery of aptamer reagents with excellent affinities and specificities, perhaps even exceeding those of the best monoclonal antibodies. Since aptamers are reproducible, renewable, stable, and can be distributed as sequence information, we anticipate that these affinity reagents will become even more valuable tools for both research and clinical applications.

Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2004-10-01

High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots. Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively. Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+.

Affinity approaches in RNAi-based therapeutics purification.

PubMed

Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

2016-05-15

The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

PubMed

Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

2010-11-01

As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.

Chimeric Antigen Receptors that Recognize Mesothelin for Cancer Immunotherapy | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the NCI have developed chimeric antigen receptors (CARs) with a high affinity for mesothelin to be used as an immunotherapy to treat pancreatic cancer, ovarian cancer, and mesothelioma. Cells that express CARs, most notably T cells, are highly reactive against their specific tumor antigen in an MHC-unrestricted manner to generate an immune response that promotes robust tumor cell elimination when infused into cancer patients.

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

PubMed Central

Liu, K H; Huang, C Y; Tsay, Y F

1999-01-01

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. PMID:10330471

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Synergistic use of compound properties and docking scores in neural network modeling of CYP2D6 binding: predicting affinity and conformational sampling.

PubMed

Bazeley, Peter S; Prithivi, Sridevi; Struble, Craig A; Povinelli, Richard J; Sem, Daniel S

2006-01-01

Cytochrome P450 2D6 (CYP2D6) is used to develop an approach for predicting affinity and relevant binding conformation(s) for highly flexible binding sites. The approach combines the use of docking scores and compound properties as attributes in building a neural network (NN) model. It begins by identifying segments of CYP2D6 that are important for binding specificity, based on structural variability among diverse CYP enzymes. A family of distinct, low-energy conformations of CYP2D6 are generated using simulated annealing (SA) and a collection of 82 compounds with known CYP2D6 affinities are docked. Interestingly, docking poses are observed on the backside of the heme as well as in the known active site. Docking scores for the active site binders, along with compound-specific attributes, are used to train a neural network model to properly bin compounds as strong binders, moderate binders, or nonbinders. Attribute selection is used to preselect the most important scores and compound-specific attributes for the model. A prediction accuracy of 85+/-6% is achieved. Dominant attributes include docking scores for three of the 20 conformations in the ensemble as well as the compound's formal charge, number of aromatic rings, and AlogP. Although compound properties were highly predictive attributes (12% improvement over baseline) in the NN-based prediction of CYP2D6 binders, their combined use with docking score attributes is synergistic (net increase of 23% above baseline). Beyond prediction of affinity, attribute selection provides a way to identify the most relevant protein conformation(s), in terms of binding competence. In the case of CYP2D6, three out of the ensemble of 20 SA-generated structures are found to be the most predictive for binding.

A model of high-affinity antibody binding to type III group B Streptococcus capsular polysaccharide.

PubMed

Wessels, M R; Muñoz, A; Kasper, D L

1987-12-01

We recently reported that the single repeating-unit pentasaccharide of type III group B Streptococcus (GBS) capsular polysaccharide is only weakly reactive with type III GBS antiserum. To further elucidate the relationship between antigen-chain length and antigenicity, tritiated oligosaccharides derived from type III capsular polysaccharide were used to generate detailed saturation binding curves with a fixed concentration of rabbit antiserum in a radioactive antigen-binding assay. A graded increase in affinity of antigen-antibody binding was seen as oligosaccharide size increased from 2.6 repeating units to 92 repeating units. These differences in affinity of antibody binding to oligosaccharides of different molecular size were confirmed by immunoprecipitation and competitive ELISA, two independent assays of antigen-antibody binding. Analysis of the saturation binding experiment indicated a difference of 300-fold in antibody-binding affinity for the largest versus the smallest tested oligosaccharides. Unexpectedly, the saturation binding values approached by the individual curves were inversely related to oligosaccharide chain length on a molar basis but equivalent on a weight basis. This observation is compatible with a model in which binding of an immunoglobulin molecule to an antigenic site on the polysaccharide facilitates subsequent binding of antibody to that antigen.

A computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis in infected cells

PubMed Central

Shen, Betty W.; Song, Yifan; Frayo, Shani; Convertine, Anthony J.; Margineantu, Daciana; Booth, Garrett; Correia, Bruno E.; Cheng, Yuanhua; Schief, William R.; Hockenbery, David M.; Press, Oliver W.; Stoddard, Barry L.; Stayton, Patrick S.; Baker, David

2014-01-01

SUMMARY Since apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homologue of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions, but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High specificity designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates. PMID:24949974

High affinity kainate receptor subunits are necessary for ionotropic but not metabotropic signaling

PubMed Central

Fernandes, Herman B.; Catches, Justin S.; Petralia, Ronald S.; Copits, Bryan A.; Xu, Jian; Russell, Theron A.; Swanson, Geoffrey T.; Contractor, Anis

2009-01-01

Summary Kainate receptors are atypical members of the glutamate receptor family which are able to signal through both ionotropic and metabotropic pathways. Of the five individual kainate receptor subunits the high-affinity subunits, GluK4 (KA1) and GluK5 (KA2), are unique in that they do not form functional homomeric receptors in recombinant expression systems, but combine with the primary subunits GluK1-3 (GluR5-7) to form heteromeric assemblies. Here we generated a GluK4 mutant mouse by disrupting the Grik4 gene locus. We found that loss of the GluK4 subunit leads to a significant reduction in synaptic kainate receptor currents. Moreover, ablation of both high-affinity subunits in GluK4/GluK5 double knockout mice leads to a complete loss of pre- and postsynaptic ionotropic function of synaptic kainate receptors. The principal subunits remain at the synaptic plasma membrane, but are distributed away from postsynaptic densities and presynaptic active zones. There is also an alteration in the properties of the remaining kainate receptors, as kainic acid application fails to elicit responses in GluK4/GluK5 knockout neurons. Despite the lack of detectable ionotropic synaptic receptors, the kainate receptor-mediated inhibition of the slow afterhyperpolarization current (IsAHP), which is dependent on metabotropic pathways, was intact in GluK4/GluK5 knockout mice. These results uncover a previously unknown critical role for the high-affinity kainate receptor subunits as obligatory components of ionotropic kainate receptor function, and further, demonstrate that kainate receptor participation in metabotropic signaling pathways does not require their classic role as ion channels. PMID:19778510

Recombinant human antibody fragment against tetanus toxoid produced by phage display

PubMed Central

Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

2014-01-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif.

PubMed

Manczyk, Noah; Yates, Bradley P; Veggiani, Gianluca; Ernst, Andreas; Sicheri, Frank; Sidhu, Sachdev S

2017-05-01

Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type. © 2017 The Protein Society.

Determination of trace glucose and forecast of human diseases by affinity adsorption solid substrate room temperature phosphorimetry based on Triticum valgaris lectin labeled with 4.0-generation dendrimers

NASA Astrophysics Data System (ADS)

Li, Zhiming; Zhu, Guohui; Liu, Jiaming; Lu, Qiaomei; Yang, Minlan; Wu, Hong; Shi, Xiumei; Chen, Xinhua

2007-08-01

A new phosphorescence labeling reagent Triton-100X-4.0G-D (4.0G-D refers to 4.0-generation dendrimers) was found. Quantitative specific affinity adsorption (AA) reaction between Triton-100X-4.0G-D-WGA and glucose (G) was carried out on the surface of nitrocellulose membrane (NCM), and the Δ Ip of the product of AA reaction was linear correlation to the content of G. Based on the facts above, a new method for the determination of trace G was established by WGA labeled with Triton-100X-4.0G-D affinity adsorption solid substrate room temperature phosphorimetry (Triton-100X-4.0G-D-WGA-AA-SS-RTP). This research showed that AA-SS-RTP for either direct method or sandwich method could combine very well the characteristics of both the high sensitivity of SS-RTP and the specificity of the AA reaction. Detection limits (LD) were 0.24 fg spot -1 for direct method and 0.18 fg spot -1 for sandwich method, indicating both of them were of high sensitivity. The method has been applied to the determination of the content of G in human serum, and the results were coincided with those obtained by glucose oxidize enzyme method. It can also be applied to forecast accurately some human diseases, such as primary hepatic carcinoma, cirrhosis, acute and chronic hepatitis, transfer hepatocellular, etc. Meanwhile, the mechanism for the determination of G with AA-SS-RTP was discussed.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Local uncontrollability for affine control systems with jumps

NASA Astrophysics Data System (ADS)

Treanţă, Savin

2017-09-01

This paper investigates affine control systems with jumps for which the ideal If(g1, …, gm) generated by the drift vector field f in the Lie algebra L(f, g1, …, gm) can be imbedded as a kernel of a linear first-order partial differential equation. It will lead us to uncontrollable affine control systems with jumps for which the corresponding reachable sets are included in explicitly described differentiable manifolds.

Natural and man-made V-gene repertoires for antibody discovery

PubMed Central

Finlay, William J. J.; Almagro, Juan C.

2012-01-01

Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety, and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process. PMID:23162556

Aptamer antagonists of myelin-derived inhibitors promote axon growth.

PubMed

Wang, Yuxuan; Khaing, Zin Z; Li, Na; Hall, Brad; Schmidt, Christine E; Ellington, Andrew D

2010-03-16

Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators.

Aptamer Antagonists of Myelin-Derived Inhibitors Promote Axon Growth

PubMed Central

Wang, Yuxuan; Khaing, Zin Z.; Li, Na; Hall, Brad; Schmidt, Christine E.; Ellington, Andrew D.

2010-01-01

Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators. PMID:20300533

From rabbit antibody repertoires to rabbit monoclonal antibodies.

PubMed

Weber, Justus; Peng, Haiyong; Rader, Christoph

2017-03-24

In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies.

Development of W-Ta generator

NASA Technical Reports Server (NTRS)

1981-01-01

This research program was used to further develop the existing W-Ta generator and to evaluate alternative adsorbents, preferably inorganic materials, as supports for the generator. During the first half year, combinations of non-complexing eluents and a variety of adsorbents, both inorganic and organic, were evaluated. Some of these adsorbents were synthetic, such as chelate resins that could be specific for tungsten. In the second half of the year, the stress was mainly on the use of complexing eluents because of the high affinity of hydrous oxides for tantalum, on the synthesis of chelate resins and on the use novel techniques (electrolytic) to solve the tantalum-adsorption problem.

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

PubMed Central

Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

2014-01-01

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

PubMed

André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

1999-11-01

Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential of dendrimers for applications as lectin-targeting device, as attested by these observations.

The fast release of sticky protons: Kinetics of substrate binding and proton release in a multidrug transporter

PubMed Central

Adam, Yoav; Tayer, Naama; Rotem, Dvir; Schreiber, Gideon; Schuldiner, Shimon

2007-01-01

EmrE is an Escherichia coli H+-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP+, a high-affinity substrate, is 2 × 107 M−1·s−1, a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the koff of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs. PMID:17984053

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

PubMed Central

Wang, R; Crawford, N M

1996-01-01

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct. PMID:8799195

Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

PubMed Central

2012-01-01

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na+-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline KM with no change in Vmax. As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling. PMID:23077721

Nonoisotopic assay for the presynaptic choline transporter reveals capacity for allosteric modulation of choline uptake.

PubMed

Ruggiero, Alicia M; Wright, Jane; Ferguson, Shawn M; Lewis, Michelle; Emerson, Katie S; Iwamoto, Hideki; Ivy, Michael T; Holmstrand, Ericka C; Ennis, Elizabeth A; Weaver, C David; Blakely, Randy D

2012-10-17

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.

Binding affinity of pro-apoptotic BH3 peptides for the anti-apoptotic Mcl-1 and A1 proteins: Molecular dynamics simulations of Mcl-1 and A1 in complex with six different BH3 peptides.

PubMed

Modi, Vivek; Sankararamakrishnan, Ramasubbu

2017-05-01

The anti-apoptotic members of Bcl-2 family of proteins bind to their pro-apoptotic counterparts to induce or prevent cell death.Based on the distinct binding profiles for specific pro-apoptotic BH3 peptides, the anti-apoptotic Bcl-2 proteins can be divided into at least two subclasses. The subclass that includes Bcl-X L binds strongly to Bad BH3 peptide while it has weak binding affinity for the second subclass of Bcl-2 proteins such as Mcl-1 and A1. Anti-apoptotic Bcl-2 proteins are considered to be attractive drug targets for anti-cancer drugs. BH3-mimetic inhibitors such as ABT-737 have been shown to be specific to Bcl-X L subclass while Mcl-1 and A1 show resistance to the same drug. An efficacious inhibitor should target all the anti-apoptotic Bcl-2 proteins. Hence, development of inhibitors selective to Mcl-1 and A1 is of prime importance for targeted cancer therapeutics. The first step to achieve this goal is to understand the molecular basis of high binding affinities of specific pro-apoptotic BH3 peptides for Mcl-1 and A1. To understand the interactions between the BH3 peptides and Mcl-1/A1, we performed multi-nanosecond molecular dynamics (MD) simulations of six complex structures of Mcl-1 and A1. With the exception of Bad, all complex structures were experimentally determined. Bad complex structures were modeled. Our simulation studies identified specific pattern of polar interactions between Mcl-1/A1 and high-affinity binding BH3 peptides. The lack of such polar interactions in Bad peptide complex is attributed to specific basic residues present before and after the highly conserved Leu residue. The close approach of basic residues in Bad and Mcl-1/A1 is hypothesized to be the cause of weak binding affinity. To test this hypothesis, we generated in silico mutants of these basic residues in Bad peptide and Mcl-1/A1 proteins. MD simulations of the mutant systems established the pattern of stable polar interactions observed in high-affinity binding BH3 peptides. We have thus identified specific residue positions in Bad and Mcl-1/A1 responsible for the weak binding affinity. Results from these simulation studies will aid in the development of inhibitors specific to Mcl-1 and A1 proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

PubMed

Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

2010-11-30

Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

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

Gao, Yun, E-mail: ygao@yorku.ca; Hu, Naihong, E-mail: nhhu@math.ecnu.edu.cn; Zhang, Honglian, E-mail: hlzhangmath@shu.edu.cn

In this paper, we define the two-parameter quantum affine algebra for type G{sub 2}{sup (1)} and give the (r, s)-Drinfeld realization of U{sub r,s}(G{sub 2}{sup (1)}), as well as establish and prove its Drinfeld isomorphism. We construct and verify explicitly the level-one vertex representation of two-parameter quantum affine algebra U{sub r,s}(G{sub 2}{sup (1)}), which also supports an evidence in nontwisted type G{sub 2}{sup (1)} for the uniform defining approach via the two-parameter τ-invariant generating functions proposed in Hu and Zhang [Generating functions with τ-invariance and vertex representations of two-parameter quantum affine algebras U{sub r,s}(g{sup ^}): Simply laced cases e-print http://arxiv.org/abs/1401.4925more » ].« less

CLUSTERING OF INTERICTAL SPIKES BY DYNAMIC TIME WARPING AND AFFINITY PROPAGATION

PubMed Central

Thomas, John; Jin, Jing; Dauwels, Justin; Cash, Sydney S.; Westover, M. Brandon

2018-01-01

Epilepsy is often associated with the presence of spikes in electroencephalograms (EEGs). The spike waveforms vary vastly among epilepsy patients, and also for the same patient across time. In order to develop semi-automated and automated methods for detecting spikes, it is crucial to obtain a better understanding of the various spike shapes. In this paper, we develop several approaches to extract exemplars of spikes. We generate spike exemplars by applying clustering algorithms to a database of spikes from 12 patients. As similarity measures for clustering, we consider the Euclidean distance and Dynamic Time Warping (DTW). We assess two clustering algorithms, namely, K-means clustering and affinity propagation. The clustering methods are compared based on the mean squared error, and the similarity measures are assessed based on the number of generated spike clusters. Affinity propagation with DTW is shown to be the best combination for clustering epileptic spikes, since it generates fewer spike templates and does not require to pre-specify the number of spike templates. PMID:29527130

Generation of Dynamic Combinatorial Libraries Using Hydrazone‐Functionalized Surface Mimetics

PubMed Central

Hewitt, Sarah H.

2018-01-01

Dynamic combinatorial chemistry (DCC) represents an approach, whereby traditional supramolecular scaffolds used for protein surface recognition might be exploited to achieve selective high affinity target recognition. Synthesis, in situ screening and amplification under selection pressure allows the generation of ligands, which bear different moieties capable of making multivalent non‐covalent interactions with target proteins. Generic tetracarboxyphenyl porphyrin scaffolds bearing four hydrazide moieties have been used to form dynamic combinatorial libraries (DCLs) using aniline‐catalyzed reversible hydrazone exchange reactions, in 10 % DMSO, 5 mm NH4OAc, at pH 6.75. High resolution mass spectrometry (HRMS) was used to monitor library composition and establish conditions under which equilibria were established.

Native CB1 receptor affinity, intrinsic activity and accumbens shell dopamine stimulant properties of third generation SPICE/K2 cannabinoids: BB-22, 5F-PB-22, 5F-AKB-48 and STS-135.

PubMed

De Luca, Maria Antonietta; Castelli, M Paola; Loi, Barbara; Porcu, Alessandra; Martorelli, Mariella; Miliano, Cristina; Kellett, Kathryn; Davidson, Colin; Stair, Jacqueline L; Schifano, Fabrizio; Di Chiara, Gaetano

2016-06-01

In order to investigate the in vivo dopamine (DA) stimulant properties of selected 3rd generation Spice/K2 cannabinoids, BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, their in vitro affinity and agonist potency at native rat and mice CB1 receptors was studied. The compounds bind with high affinity to CB1 receptors in rat cerebral cortex homogenates and stimulate CB1-induced [(35)S]GTPγS binding with high potency and efficacy. BB-22 and 5F-PB-22 showed the lowest Ki of binding to CB1 receptors (0.11 and 0.13 nM), i.e., 30 and 26 times lower respectively than that of JWH-018 (3.38 nM), and a potency (EC50, 2.9 and 3.7 nM, respectively) and efficacy (Emax, 217% and 203%, respectively) as CB1 agonists higher than JWH-018 (EC50, 20.2 nM; Emax, 163%). 5F-AKB-48 and STS-135 had higher Ki for CB1 binding, higher EC50 and lower Emax as CB1 agonists than BB-22 and 5F-PB-22 but still comparatively more favourable than JWH-018. The agonist properties of all the compounds were abolished or drastically reduced by the CB1 antagonist/inverse agonist AM251 (0.1 μM). No activation of G-protein was observed in CB1-KO mice. BB-22 (0.003-0.01 mg/kg i.v.) increased dialysate DA in the accumbens shell but not in the core or in the medial prefrontal cortex, with a bell shaped dose-response curve and an effect at 0.01 mg/kg and a biphasic time-course. Systemic AM251 (1.0 mg/kg i.p.) completely prevented the stimulant effect of BB-22 on dialysate DA in the NAc shell. All the other compounds increased dialysate DA in the NAc shell at doses consistent with their in vitro affinity for CB1 receptors (5F-PB-22, 0.01 mg/kg; 5F-AKB-48, 0.1 mg/kg; STS-135, 0.15 mg/kg i.v.). 3rd generation cannabinoids can be even more potent and super-high CB1 receptor agonists compared to JWH-018. Future research will try to establish if these properties can explain the high toxicity and lethality associated with these compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Wurzburg, Beth A.; Kim, Beomkyu; Tarchevskaya, Svetlana S.

IgE antibodies interact with the high affinity IgE Fc receptor, FcϵRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcϵRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of anmore » IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcϵRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.« less

Three-dimensional quantitative structure-activity relationship modeling of cocaine binding by a novel human monoclonal antibody.

PubMed

Paula, Stefan; Tabet, Michael R; Farr, Carol D; Norman, Andrew B; Ball, W James

2004-01-01

Human monoclonal antibodies (mAbs) designed for immunotherapy have a high potential for avoiding the complications that may result from human immune system responses to the introduction of nonhuman mAbs into patients. This study presents a characterization of cocaine/antibody interactions that determine the binding properties of the novel human sequence mAb 2E2 using three-dimensional quantitative structure-activity relationship (3D-QSAR) methodology. We have experimentally determined the binding affinities of mAb 2E2 for cocaine and 38 cocaine analogues. The K(d) of mAb 2E2 for cocaine was 4 nM, indicating a high affinity. Also, mAb 2E2 displayed good cocaine specificity, as reflected in its 10-, 1500-, and 25000-fold lower binding affinities for the three physiologically relevant cocaine metabolites benzoylecgonine, ecgonine methyl ester, and ecgonine, respectively. 3D-QSAR models of cocaine binding were developed by comparative molecular similarity index analysis (CoMSIA). A model of high statistical quality was generated showing that cocaine binds to mAb 2E2 in a sterically restricted binding site that leaves the methyl group attached to the ring nitrogen of cocaine solvent-exposed. The methyl ester group of cocaine appears to engage in attractive van der Waals interactions with mAb 2E2, whereas the phenyl group contributes to the binding primarily via hydrophobic interactions. The model further indicated that an increase in partial positive charge near the nitrogen proton and methyl ester carbonyl group enhances binding affinity and that the ester oxygen likely forms an intermolecular hydrogen bond with mAb 2E2. Overall, the cocaine binding properties of mAb 2E2 support its clinical potential for development as a treatment of cocaine overdose and addiction.

Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA*

PubMed Central

Schoenherr, Regine M.; Saul, Richard G.; Whiteaker, Jeffrey R.; Yan, Ping; Whiteley, Gordon R.; Paulovich, Amanda G.

2015-01-01

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach. PMID:25512614

Design and Development of a Technology Platform for DNA-Encoded Library Production and Affinity Selection.

PubMed

Castañón, Jesús; Román, José Pablo; Jessop, Theodore C; de Blas, Jesús; Haro, Rubén

2018-06-01

DNA-encoded libraries (DELs) have emerged as an efficient and cost-effective drug discovery tool for the exploration and screening of very large chemical space using small-molecule collections of unprecedented size. Herein, we report an integrated automation and informatics system designed to enhance the quality, efficiency, and throughput of the production and affinity selection of these libraries. The platform is governed by software developed according to a database-centric architecture to ensure data consistency, integrity, and availability. Through its versatile protocol management functionalities, this application captures the wide diversity of experimental processes involved with DEL technology, keeps track of working protocols in the database, and uses them to command robotic liquid handlers for the synthesis of libraries. This approach provides full traceability of building-blocks and DNA tags in each split-and-pool cycle. Affinity selection experiments and high-throughput sequencing reads are also captured in the database, and the results are automatically deconvoluted and visualized in customizable representations. Researchers can compare results of different experiments and use machine learning methods to discover patterns in data. As of this writing, the platform has been validated through the generation and affinity selection of various libraries, and it has become the cornerstone of the DEL production effort at Lilly.

Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization

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

Sogabe, Satoshi; Sakamoto, Kotaro; Kamada, Yusuke

Keap1 constitutively binds to the transcription factor Nrf2 to promote its degradation, resulting in negative modulation of genes involved in cellular protection against oxidative stress. Keap1 is increasingly recognized as an attractive target for treating diseases involving oxidative stress, including cancer, atherosclerosis, diabetes, arthritis, and neurodegeneration. We used phage-display peptide screening to identify a tetrapeptide showing moderate binding affinity, which inhibits the interaction between Nrf2 and Keap1. The tetrapeptide does not include an ETGE motif, which is a commonly found consensus sequence in known peptidic inhibitors. In addition to affinity parameters, IC{sub 50}, K{sub D}, and thermodynamic parameters, the crystalmore » structure of the complex was determined to elucidate the binding conformation. The binding interactions resemble those of known small-molecule inhibitors as opposed to those of substrates and peptidic inhibitors. Although the tetrapeptide's affinity is not very high, our results may help facilitate the designing of small-molecule inhibitors during lead generation in drug discovery. - Highlights: • Keap1 inhibitory tetrapeptide with moderate affinity was discovered. • Crystal structure of the complex showed the unique binding mode. • Structural information gives a valuable insight for design of therapeutic compounds.« less

On the connection between inherent DNA flexure and preferred binding of hydroxymethyluracil-containing DNA by the type II DNA-binding protein TF1.

PubMed

Grove, A; Galeone, A; Mayol, L; Geiduschek, E P

1996-07-12

TF1 is a member of the family of type II DNA-binding proteins, which also includes the bacterial HU proteins and the Escherichia coli integration host factor (IHF). Distinctive to TF1, which is encoded by the Bacillus subtilis bacteriophage SPO1, is its preferential binding to DNA in which thymine is replaced by 5-hydroxymethyluracil (hmU), as it is in the phage genome. TF1 binds to preferred sites within the phage genome and generates pronounced DNA bending. The extent to which DNA flexibility contributes to the sequence-specific binding of TF1, and the connection between hmU preference and DNA flexibility has been examined. Model flexible sites, consisting of consecutive mismatches, increase the affinity of thymine-containing DNA for TF1. In particular, tandem mismatches separated by nine base-pairs generate an increase, by orders of magnitude, in the affinity of TF1 for T-containing DNA with the sequence of a preferred TF1 binding site, and fully match the affinity of TF1 for this cognate site in hmU-containing DNA (Kd approximately 3 nM). Other placements of loops generate suboptimal binding. This is consistent with a significant contribution of site-specific DNA flexibility to complex formation. Analysis of complexes with hmU-DNA of decreasing length shows that a major part of the binding affinity is generated within a central 19 bp segment (delta G0 = 41.7 kJ mol-1) with more-distal DNA contributing modestly to the affinity (delta delta G = -0.42 kJ mol-1 bp-1 on increasing duplex length to 37 bp). However, a previously characterised thermostable and more tightly binding mutant TF1, TF1(E15G/T32I), derives most of its extra affinity from interaction with flanking DNA. We propose that inherent but sequence-dependent deformability of hmU-containing DNA underlies the preferential binding of TF1 and that TF1-induced DNA bendings is a result of distortions at two distinct sites separated by 9 bp of duplex DNA.

Impact of antipsychotic medication on transcranial direct current stimulation (tDCS) effects in schizophrenia patients.

PubMed

Agarwal, Sri Mahavir; Bose, Anushree; Shivakumar, Venkataram; Narayanaswamy, Janardhanan C; Chhabra, Harleen; Kalmady, Sunil V; Varambally, Shivarama; Nitsche, Michael A; Venkatasubramanian, Ganesan; Gangadhar, Bangalore N

2016-01-30

Transcranial direct current stimulation (tDCS) has generated interest as a treatment modality for schizophrenia. Dopamine, a critical pathogenetic link in schizophrenia, is also known to influence tDCS effects. We evaluated the influence of antipsychotic drug type (as defined by dopamine D2 receptor affinity) on the impact of tDCS in schizophrenia. DSM-IV-TR-diagnosed schizophrenia patients [N=36] with persistent auditory hallucinations despite adequate antipsychotic treatment were administered add-on tDCS. Patients were divided into three groups based on the antipsychotic's affinity to D2 receptors. An auditory hallucinations score (AHS) was measured using the auditory hallucinations subscale of the Psychotic Symptom Rating Scales (PSYRATS). Add-on tDCS resulted in a significant reduction inAHS. Antipsychotic drug type had a significant effect on AHS reduction. Patients treated with high affinity antipsychotics showed significantly lesser improvement compared to patients on low affinity antipsychotics or a mixture of the two. Furthermore, a significant sex-by-group interaction occurred; type of medication had an impact on tDCS effects only in women. Improvement differences could be due to the larger availability of the dopamine receptor system in patients taking antipsychotics with low D2 affinity. Sex-specific differences suggest potential estrogen-mediated effects. This study reports a first-time observation on the clinical utility of antipsychotic drug type in predicting tDCS effects in schizophrenia. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Scoring functions for protein-protein interactions.

PubMed

Moal, Iain H; Moretti, Rocco; Baker, David; Fernández-Recio, Juan

2013-12-01

The computational evaluation of protein-protein interactions will play an important role in organising the wealth of data being generated by high-throughput initiatives. Here we discuss future applications, report recent developments and identify areas requiring further investigation. Many functions have been developed to quantify the structural and energetic properties of interacting proteins, finding use in interrelated challenges revolving around the relationship between sequence, structure and binding free energy. These include loop modelling, side-chain refinement, docking, multimer assembly, affinity prediction, affinity change upon mutation, hotspots location and interface design. Information derived from models optimised for one of these challenges can be used to benefit the others, and can be unified within the theoretical frameworks of multi-task learning and Pareto-optimal multi-objective learning. Copyright © 2013 Elsevier Ltd. All rights reserved.

Expression and Interaction Analysis among Saffron ALDHs and Crocetin Dialdehyde.

PubMed

Gómez-Gómez, Lourdes; Pacios, Luis F; Diaz-Perales, Araceli; Garrido-Arandia, María; Argandoña, Javier; Rubio-Moraga, Ángela; Ahrazem, Oussama

2018-05-09

In saffron, the cleavage of zeaxanthin by means of CCD2 generates crocetin dialdehyde, which is then converted by an unknown aldehyde dehydrogenase to crocetin. A proteome from saffron stigma was released recently and, based on the expression pattern and correlation analyses, five aldehyde dehydrogenases (ALDHs) were suggested as possible candidates to generate crocetin from crocetin dialdehydes. We selected four of the suggested ALDHs and analyzed their expression in different tissues, determined their activity over crocetin dialdehyde, and performed structure modeling and docking calculation to find their specificity. All the ALDHs were able to convert crocetin dialdehyde to crocetin, but two of them were stigma tissue-specific. Structure modeling and docking analyses revealed that, in all cases, there was a high coverage of residues in the models. All of them showed a very close conformation, indicated by the low root-mean-square deviation (RMSD) values of backbone atoms, which indicate a high similarity among them. However, low affinity between the enzymes and the crocetin dialdehyde were observed. Phylogenetic analysis and binding affinities calculations, including some ALDHs from Gardenia jasmonoides , Crocus sieberi , and Buddleja species that accumulate crocetin and Bixa orellana synthetizing the apocarotenoid bixin selected on their expression pattern matching with the accumulation of either crocins or bixin, pointed out that family 2 C4 members might be involved in the conversion of crocetin dialdehyde to crocetin with high specificity.

[Interaction of human factor X with thromboplastin].

PubMed

Kiselev, S V; Zubairov, D M; Timarbaev, V N

2003-01-01

The binding of 125I-labeled human factor X to native and papaine-treated tissue tromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard analysis suggests the existence of high (Kd=l,8 x10(-9) M) and low affinity binding sites on the thromboplastin surface. The removal of Ca2+ reduced affinity of factor X to the high affinity sites. This was accompanied by some increase of their number. Proteolysis by papaine decreased affinity of high affinity sites and caused the increase of their number in the presence of Ca2+. In the absence of Ca2+ the affinity remained unchanged, but the number of sites decreased. At low concentrations of factor X positive cooperativity for high affinity binding sites was observed. It did not depend on the presence of Ca2+. The results indirectly confirm the role of hydrophobic interactons in Ca2+ dependent binding of factor X to thromboplastin and the fact that heterogeneity of this binding is determined by mesophase structure of the thromboplastin phospholipids.

Torque Generation Mechanism of F1-ATPase upon NTP Binding

PubMed Central

Arai, Hidenobu C.; Yukawa, Ayako; Iwatate, Ryu John; Kamiya, Mako; Watanabe, Rikiya; Urano, Yasuteru; Noji, Hiroyuki

2014-01-01

Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F1-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 103 and 106 times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F1 to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F1 exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F1 with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity. PMID:24988350

Construction of Rabbit Immune Antibody Libraries.

PubMed

Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

2018-01-01

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Insight into the Meligethes aeneus voltage-sensitive sodium channel structure and an attempt to select the best pyrethroid ligands.

PubMed

Obrępalska-Stęplowska, Aleksandra; Czerwoniec, Anna; Wieczorek, Przemysław; Wrzesińska, Barbara

2016-01-01

The voltage-sensitive sodium channel (VSSC) is a target for the pharmacological action of pyrethroids which are used in controlling pests, including those of agricultural importance. Among these is the pollen beetle (Meligethes aeneus F.) - the most serious pest of Brassica napus. Owing to the heavy use of pyrethroids, a widespread build-up of resistance has occurred. The main cause of pyrethroid insensitivity in M. aeneus is considered to be an increased oxidative metabolism; however, the additional mechanism of resistance associated with mutations in the VSSC might contribute to this phenomenon. We generated a VSSC 3D model to study the docking affinities of pyrethroids to their target site within the channel. Our goal was to identify the pyrethroids for which docking affinity scores were high and not affected by potential mutations in the VSSC. We found that the docking scores of cypermethrin are hardly influenced by the appearance of point mutations. Additionally, tau-fluvalinate, deltamethrin and bifenthrin are VSSC ligands with high affinity scores. Our docking models suggest that point mutations in the VSSC binding pocket might affect the stability of ligand interactions and change the pattern of ligand docking locations, which might have a potential effect on VSSC gating properties. © 2015 Society of Chemical Industry.

Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.

PubMed

Malm, Magdalena; Kronqvist, Nina; Lindberg, Hanna; Gudmundsdotter, Lindvi; Bass, Tarek; Frejd, Fredrik Y; Höidén-Guthenberg, Ingmarie; Varasteh, Zohreh; Orlova, Anna; Tolmachev, Vladimir; Ståhl, Stefan; Löfblom, John

2013-01-01

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

Thermodynamic Bounds on the Ultra- and Infra-affinity of Hsp70 for Its Substrates

NASA Astrophysics Data System (ADS)

Nguyen, Basile; Hartich, David; Seifert, Udo; Rios, Paolo De Los

2017-07-01

The 70 kDa Heat Shock Proteins Hsp70 have several essential functions in living systems, such as protecting cells against protein aggregation, assisting protein folding, remodeling protein complexes and driving the translocation into organelles. These functions require high affinity for non-specific amino-acid sequences that are ubiquitous in proteins. It has been recently shown that this high affinity, called ultra-affinity, depends on a process driven out of equilibrium by ATP hydrolysis. Here we establish the thermodynamic bounds for ultra-affinity, and further show that the same reaction scheme can in principle be used both to strengthen and to weaken affinities (leading in this case to infra-affinity). We show that cofactors are essential to achieve affinity beyond the equilibrium range. Finally, biological implications are discussed.

WORKSHOP ON DEVELOPMENT OF RADIONUCLIDE GETTERS FOR THE YUCCA MOUNTAIN WASTE REPOSITORY

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

K.C. Holt

One of the important that the U.S. Department of Energy (DOE) is currently undertaking is the development of a high-level nuclear waste repository to be located at Yucca Mountain, Nevada. Concern is generated by the Yucca Mountain Project (YMP) is due to potential releases as groundwater contamination, as described in the Total System Performance Assessment (TSPA). The dose to an off-site individual using this groundwater for drinking and irrigation is dominated by four radionuclides: Tc-99, I-127, Np-237, and U-238. Ideally, this dose would be limited to a single radionuclide, U-238; in other words, YMP would resemble a uranium ore body,more » a common geologic feature in the Western U.S. For this reason and because of uncertainties in the behavior of Tc-99, I-127, and Np-237, it would be helpful to limit the amount of Tc, I, and Np leaving the repository, which would greatly increase the confidence in the long-term performance of YMP. An approach to limiting the migration of Tc, I, and Np that is complementary to the existing YMP repository design plans is to employ sequestering agents or ''getters'' for these radionuclides such that their migration is greatly hindered, thus decreasing the amount of radionuclide leaving the repository. Development of such getters presents a number of significant challenges. The getter must have a high affinity and high selectivity for the radionuclide in question since there is approximately a 20- to 50-fold excess of other fission products and a 1000-fold excess of uranium in addition to the ions present in the groundwater. An even greater challenge is that the getters must function over a period greater than the half-life of the radionuclide (greater than 5 half-lives would be ideal). Typically, materials with a high affinity for Tc, I, or Np are not sufficiently durable. For example, strong-base ion exchange resins have a very high affinity for TcO{sub 4}{sup -} but are not expected to be durable. On the other hand, durable materials, such as hydrotalcite, do not have sufficient affinity to be useful getters. Despite these problems, the great increase in the repository performance and corresponding decrease in uncertainty promised by a useful getter has generated significant interest in these materials. This report is the result a workshop sponsored by the Office of Civilian Radioactive Waste Management and Office of Science and Technology and International of the DOE to assess the state of research in this field.« less

Development of a fraction collection approach in capillary electrophoresis SELEX for aptamer selection.

PubMed

Luo, Zhaofeng; Zhou, Hongmin; Jiang, Hao; Ou, Huichao; Li, Xin; Zhang, Liyun

2015-04-21

Aptamers have attracted much attention due to their ability to bind to target molecules with high affinity and specificity. The development of an approach capable of efficiently generating aptamers through systematic evolution of ligands by exponential enrichment (SELEX) is particularly challenging. Herein, a fraction collection approach in capillary electrophoresis SELEX (FCE-SELEX) for the partition of a bound DNA-target complex is developed. By integrating fraction collection with a facile oil seal method for avoiding contamination while amplifying the bound DNA-target complex, in a single round of selection, a streptavidin-binding aptamer (SBA) has been generated. The affinity of aptamer SBA-36 for streptavidin (SA) is determined as 30.8 nM by surface plasmon resonance (SPR). Selectivity and biotin competition experiments demonstrate that the SBA-36 aptamer selected by FCE-SELEX is as efficient as those from other methods. Based on the ability of fraction collection in partition and collection of the aptamer-target complex from the original DNA library, FCE-SELEX can be a universal tool for the development of aptamers.

Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.

PubMed

Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge

2012-01-01

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

High ionic strength depresses muscle contractility by decreasing both force per cross-bridge and the number of strongly attached cross-bridges.

PubMed

Wang, Li; Bahadir, Anzel; Kawai, Masataka

2015-06-01

An increase in ionic strength (IS) lowers Ca(2+) activated tension in muscle fibres, however, its molecular mechanism is not well understood. In this study, we used single rabbit psoas fibres to perform sinusoidal analyses. During Ca(2+) activation, the effects of ligands (ATP, Pi, and ADP) at IS ranging 150-300 mM were studied on three rate constants to characterize elementary steps of the cross-bridge cycle. The IS effects were studied because a change in IS modifies the inter- and intra-molecular interactions, hence they may shed light on the molecular mechanisms of force generation. Both the ATP binding affinity (K1) and the ADP binding affinity (K 0) increased to 2-3x, and the Pi binding affinity (K5) decreased to 1/2, when IS was raised from 150 to 300 mM. The effect on ATP/ADP can be explained by stereospecific and hydrophobic interaction, and the effect on Pi can be explained by the electrostatic interaction with myosin. The increase in IS increased cross-bridge detachment steps (k2 and k-4), indicating that electrostatic repulsion promotes these steps. However, IS did not affect attachment steps (k-2 and k4). Consequently, the equilibrium constant of the detachment step (K2) increased by ~100%, and the force generation step (K4) decreased by ~30%. These effects together diminished the number of force-generating cross-bridges by 11%. Force/cross-bridge (T56) decreased by 26%, which correlates well with a decrease in the Debye length that limits the ionic atmosphere where ionic interactions take place. We conclude that the major effect of IS is a decrease in force/cross-bridge, but a decrease in the number of force generating cross-bridge also takes place. The stiffness during rigor induction did not change with IS, demonstrating that in-series compliance is not much affected by IS.

High Ionic Strength Depresses Muscle Contractility by Decreasing both Force per Cross-bridge and the Number of Strongly Attached Cross-bridges

PubMed Central

Wang, Li; Bahadir, Anzel; Kawai, Masataka

2015-01-01

An increase in ionic strength (IS) lowers Ca2+ activated tension in muscle fibres, however, its molecular mechanism is not well understood. In this study, we used single rabbit psoas fibres to perform sinusoidal analyses. During Ca2+ activation, the effects of ligands (ATP, Pi, and ADP) at IS ranging 150 mM – 300 mM were studied on three rate constants to characterize elementary steps of the cross-bridge cycle. The IS effects were studied because a change in IS modifies the inter- and intra-molecular interactions, hence they may shed light on the molecular mechanisms of force generation. Both the ATP binding affinity (K1) and the ADP binding affinity (K0) increased to 2-3x, and the Pi binding affinity (K5) decreased to 1/2, when IS was raised from 150 mM to 300 mM. The effect on ATP/ADP can be explained by stereospecific and hydrophobic interaction, and the effect on Pi can be explained by the electrostatic interaction with myosin. The increase in IS increased cross-bridge detachment steps (k2 and k−4), indicating that electrostatic repulsion promotes these steps. However, IS did not affect attachment steps (k−2 and k4). Consequently, the equilibrium constant of the detachment step (K2) increased by ~100%, and the force generation step (K4) decreased by ~30%. These effects together diminished the number of force-generating cross-bridges by 11%. Force/cross-bridge (T56) decreased by 26%, which correlates well with a decrease in the Debye length that limits the ionic atmosphere where ionic interactions take place. We conclude that the major effect of IS is a decrease in force/cross-bridge, but a decrease in the number of force generating cross-bridge also takes place. The stiffness during rigor induction did not change with IS, demonstrating that in-series compliance is not much affected by IS. PMID:25836331

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

PubMed Central

Nampally, Malathi; Moerschbacher, Bruno Maria

2012-01-01

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

A study of the uptake of chloroquine in malaria-infected erythrocytes. High and low affinity uptake and the influence of glucose and its analogues.

PubMed

Diribe, C O; Warhurst, D C

1985-09-01

A study of concentration- and substrate-dependence of chloroquine uptake has been carried out on mouse erythrocytes infected with the chloroquine-sensitive NK65 and the chloroquine-resistant RC strains of Plasmodium berghei. The presence of drug binding sites of high and low affinity in such strains of P. berghei was confirmed. High affinity uptake sites in cells parasitized with chloroquine-sensitive and chloroquine-resistant parasites have similar characteristics, but in the sensitive strain the major component of chloroquine-uptake is at high affinity and dependent on the availability of ATP whilst in the resistant strain the major component of uptake is at low affinity and independent of energy. An absolute increase in the quantity of the low affinity site in erythrocytes parasitized with chloroquine-resistant P. berghei was noted, which may be related to an increase in quantity of parasite membrane.

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

Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji

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

Investigation of the Mechanism of Electron Capture and Electron Transfer Dissociation of Peptides with a Covalently Attached Free Radical Hydrogen Atom Scavenger.

PubMed

Sohn, Chang Ho; Yin, Sheng; Peng, Ivory; Loo, Joseph A; Beauchamp, J L

2015-11-15

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280 kJ/mol) and low electron affinity (ca. 0.45 ev), and was derivatized to the model peptide, FQX TEMPO EEQQQTEDELQDK. The X TEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882 kJ/mol) of TEMPO is similar to backbone amide carbonyls (889 kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision induced dissociation (CID) of the TEMPO tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of N-C α backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO tagged peptide dication produced a series of b + H (b H ) and y + H (y H ) ions along with some c ions having suppressed intensities, consistent with stable O-H bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.

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

PubMed Central

2013-01-01

Background The cellular function of a vast majority of proteins is performed through physical interactions with other biomolecules, which, most of the time, are other proteins. Peptides represent templates of choice for mimicking a secondary structure in order to modulate protein-protein interaction. They are thus an interesting class of therapeutics since they also display strong activity, high selectivity, low toxicity and few drug-drug interactions. Furthermore, predicting peptides that would bind to a specific MHC alleles would be of tremendous benefit to improve vaccine based therapy and possibly generate antibodies with greater affinity. Modern computational methods have the potential to accelerate and lower the cost of drug and vaccine discovery by selecting potential compounds for testing in silico prior to biological validation. Results We propose a specialized string kernel for small bio-molecules, peptides and pseudo-sequences of binding interfaces. The kernel incorporates physico-chemical properties of amino acids and elegantly generalizes eight kernels, comprised of the Oligo, the Weighted Degree, the Blended Spectrum, and the Radial Basis Function. We provide a low complexity dynamic programming algorithm for the exact computation of the kernel and a linear time algorithm for it’s approximation. Combined with kernel ridge regression and SupCK, a novel binding pocket kernel, the proposed kernel yields biologically relevant and good prediction accuracy on the PepX database. For the first time, a machine learning predictor is capable of predicting the binding affinity of any peptide to any protein with reasonable accuracy. The method was also applied to both single-target and pan-specific Major Histocompatibility Complex class II benchmark datasets and three Quantitative Structure Affinity Model benchmark datasets. Conclusion On all benchmarks, our method significantly (p-value ≤ 0.057) outperforms the current state-of-the-art methods at predicting peptide-protein binding affinities. The proposed approach is flexible and can be applied to predict any quantitative biological activity. Moreover, generating reliable peptide-protein binding affinities will also improve system biology modelling of interaction pathways. Lastly, the method should be of value to a large segment of the research community with the potential to accelerate the discovery of peptide-based drugs and facilitate vaccine development. The proposed kernel is freely available at http://graal.ift.ulaval.ca/downloads/gs-kernel/. PMID:23497081

Slowing Translation between Protein Domains by Increasing Affinity between mRNAs and the Ribosomal Anti-Shine-Dalgarno Sequence Improves Solubility.

PubMed

Vasquez, Kevin A; Hatridge, Taylor A; Curtis, Nicholas C; Contreras, Lydia M

2016-02-19

Recent studies have demonstrated that effective protein production requires coordination of multiple cotranslational cellular processes, which are heavily affected by translation timing. Until recently, protein engineering has focused on codon optimization to maximize protein production rates, mostly considering the effect of tRNA abundance. However, as it relates to complex multidomain proteins, it has been hypothesized that strategic translational pauses between domains and between distinct individual structural motifs can prevent interactions between nascent chain fragments that generate kinetically trapped misfolded peptides and thereby enhance protein yields. In this study, we introduce synthetic transient pauses between structural domains in a heterologous model protein based on designed patterns of affinity between the mRNA and the anti-Shine-Dalgarno (aSD) sequence on the ribosome. We demonstrate that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria. Exploration of the affinity space showed that modifying less than 1% of the nucleotides (on a small 12 amino acid linker) can vary soluble protein yields up to ∼7-fold without altering the primary sequence of the protein. In the context of longer linkers, where a larger number of distinct structural motifs can fold outside the ribosome, optimal synonymous codon variations resulted in an additional 2.1-fold increase in solubility, relative to that of nonoptimized linkers of the same length. While rational construction of 54 linkers of various affinities showed a significant correlation between protein solubility and predicted affinity, only weaker correlations were observed between tRNA abundance and protein solubility. We also demonstrate that naturally occurring high-affinity clusters are present between structural domains of β-galactosidase, one of Escherichia coli's largest native proteins. Interdomain ribosomal affinity is an important factor that has not previously been explored in the context of protein engineering.

Affine group formulation of the Standard Model coupled to gravity

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

Chou, Ching-Yi, E-mail: l2897107@mail.ncku.edu.tw; Ita, Eyo, E-mail: ita@usna.edu; Soo, Chopin, E-mail: cpsoo@mail.ncku.edu.tw

In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of themore » Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.« less

A theory of germinal center B cell selection, division, and exit.

PubMed

Meyer-Hermann, Michael; Mohr, Elodie; Pelletier, Nadége; Zhang, Yang; Victora, Gabriel D; Toellner, Kai-Michael

2012-07-26

High-affinity antibodies are generated in germinal centers in a process involving mutation and selection of B cells. Information processing in germinal center reactions has been investigated in a number of recent experiments. These have revealed cell migration patterns, asymmetric cell divisions, and cell-cell interaction characteristics, used here to develop a theory of germinal center B cell selection, division, and exit (the LEDA model). According to this model, B cells selected by T follicular helper cells on the basis of successful antigen processing always return to the dark zone for asymmetric division, and acquired antigen is inherited by one daughter cell only. Antigen-retaining B cells differentiate to plasma cells and leave the germinal center through the dark zone. This theory has implications for the functioning of germinal centers because compared to previous models, high-affinity antibodies appear one day earlier and the amount of derived plasma cells is considerably larger. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Localisation of the high-affinity choline transporter-1 in the rat skeletal motor unit.

PubMed

Lips, Katrin S; Pfeil, Uwe; Haberberger, Rainer V; Kummer, Wolfgang

2002-03-01

The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled alpha-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.

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

Pennington, Luke F.; Tarchevskaya, Svetlana; Brigger, Daniel

Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab–Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, inmore » combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. Furthermore, this combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions.« less

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments

ERIC Educational Resources Information Center

Bailey, Cheryl P.

2009-01-01

This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations…

Problems and Limitations of Satellite Image Orientation for Determination of Height Models

NASA Astrophysics Data System (ADS)

Jacobsen, K.

2017-05-01

The usual satellite image orientation is based on bias corrected rational polynomial coefficients (RPC). The RPC are describing the direct sensor orientation of the satellite images. The locations of the projection centres today are without problems, but an accuracy limit is caused by the attitudes. Very high resolution satellites today are very agile, able to change the pointed area over 200km within 10 to 11 seconds. The corresponding fast attitude acceleration of the satellite may cause a jitter which cannot be expressed by the third order RPC, even if it is recorded by the gyros. Only a correction of the image geometry may help, but usually this will not be done. The first indication of jitter problems is shown by systematic errors of the y-parallaxes (py) for the intersection of corresponding points during the computation of ground coordinates. These y-parallaxes have a limited influence to the ground coordinates, but similar problems can be expected for the x-parallaxes, determining directly the object height. Systematic y-parallaxes are shown for Ziyuan-3 (ZY3), WorldView-2 (WV2), Pleiades, Cartosat-1, IKONOS and GeoEye. Some of them have clear jitter effects. In addition linear trends of py can be seen. Linear trends in py and tilts in of computed height models may be caused by limited accuracy of the attitude registration, but also by bias correction with affinity transformation. The bias correction is based on ground control points (GCPs). The accuracy of the GCPs usually does not cause some limitations but the identification of the GCPs in the images may be difficult. With 2-dimensional bias corrected RPC-orientation by affinity transformation tilts of the generated height models may be caused, but due to large affine image deformations some satellites, as Cartosat-1, have to be handled with bias correction by affinity transformation. Instead of a 2-dimensional RPC-orientation also a 3-dimensional orientation is possible, respecting the object height more as by 2-dimensional orientation. The 3-dimensional orientation showed advantages for orientation based on a limited number of GCPs, but in case of poor GCP distribution it may cause also negative effects. For some of the used satellites the bias correction by affinity transformation showed advantages, but for some other the bias correction by shift was leading to a better levelling of the generated height models, even if the root mean square (RMS) differences at the GCPs were larger as for bias correction by affinity transformation. The generated height models can be analyzed and corrected with reference height models. For the used data sets accurate reference height models are available, but an analysis and correction with the free of charge available SRTM digital surface model (DSM) or ALOS World 3D (AW3D30) is also possible and leads to similar results. The comparison of the generated height models with the reference DSM shows some height undulations, but the major accuracy influence is caused by tilts of the height models. Some height model undulations reach up to 50 % of the ground sampling distance (GSD), this is not negligible but it cannot be seen not so much at the standard deviations of the height. In any case an improvement of the generated height models is possible with reference height models. If such corrections are applied it compensates possible negative effects of the type of bias correction or 2-dimensional orientations against 3-dimensional handling.

[Role of hemoglobin affinity to oxygen in adaptation to hypoxemia].

PubMed

Kwasiborski, Przemysław Jerzy; Kowalczyk, Paweł; Zieliński, Jakub; Przybylski, Jacek; Cwetsch, Andrzej

2010-04-01

One of the basic mechanisms of adapting to hypoxemia is a decrease in the affinity of hemoglobin for oxygen. This process occurs mainly due to the increased synthesis of 2,3-diphosphoglycerate (2,3-DPG) in the erythrocytes, as well as through the Bohr effect. Hemoglobin with decreased affinity for oxygen increases the oxygenation of tissues, because it gives up oxygen more easily during microcirculation. In foetal circulation, however, at a partial oxygen pressure (pO2) of 25 mmHg in the umbilical vein, the oxygen carrier is type F hemoglobin which has a high oxygen affinity. The commonly accepted role for hemoglobin F is limited to facilitating diffusion through the placenta. Is fetal life the only moment when haemoglobin F is useful? THE AIM OF STUDY was to create a mathematical model, which would answer the question at what conditions an increase, rather than a decrease, in haemoglobin oxygen affinity is of benefit to the body. Using the kinetics of dissociation of oxygen from hemoglobin described by the Hill equation as the basis for further discussion, we created a mathematical model describing the pO2 value in the microcirculatory system and its dependence on arterial blood pO2. The calculations were performed for hemoglobin with low oxygen affinity (adult type) and high-affinity hemoglobin (fetal type). The modelling took into account both physiological and pathological ranges of acid-base equilibrium and tissue oxygen extraction parameters. It was shown that for the physiological range of acid-base equilibrium and the resting level of tissue oxygen extraction parameters, with an arterial blood pO2 of 26.8 mmHg, the higher-affinity hemoglobin becomes the more effective oxygen carrier. It was also demonstrated that the arterial blood pO2, below which the high-affinity hemoglobin becomes the more effective carrier, is dependent on blood pH and the difference between the arterial and venous oxygen saturation levels. Simulations performed for the pathological states showed that acidosis and increased tissue oxygen demand lead to a broadened arterial blood pO2 range, in which the high-affinity hemoglobin is more efficient. Contrary to the widely held view that the only response to hypoxemia is a decrease in haemoglobin oxygen affinity, it was shown that under extreme hypoxemic conditions, an increased haemoglobin oxygen affinity improves the oxygenation of tissues. It was also shown that the dominance of hemoglobin with a high oxygen affinity rapidly exceeds hemoglobin with low oxygen affinity in the case of acidosis with its accompanying high tissue oxygen extraction. In cases of extreme disruptions of the acid-base equilibrium, the dominance of high-oxygen-affinity hemoglobin spans over the entire possible range of pO2 in arterial blood.

[Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell].

PubMed

Tan, Yan; Liang, Huiyu; Wu, Xidong; Gao, Yubo; Zhang, Xingmei

2013-05-01

A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.

Thiolation mediated pegylation platform to generate functional universal red blood cells.

PubMed

Nacharaju, Parimala; Manjula, Belur N; Acharya, Seetharama A

2007-01-01

The PEGylation that adds an extension arm on protein amino groups with the conservation of their positive charge masks the A and D antigens of erythrocytes efficiently. In the present study, the efficiency of masking the antigens of RBC by PEGylation protocols that do not conserve the charge with and without adding extension arms is compared. The conjugation of PEG-5000 to RBCs through the addition of extension arms masked the D antigen more efficiently than the other protocol. A combination of PEG-5 K and PEG-20 K is needed to mask the A antigen, irrespective of the PEGylation approach. The oxygen affinity of the PEGylated RBCs increased by the extension arm facilitated PEGylation. The protocol involving the conjugation of PEG-chains without adding extension arm did not alter the oxygen affinity of RBCs. A combination of PEGylation protocols is an alternate strategy to generate universal red blood cells with good levels of oxygen affinity.

Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

PubMed Central

LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

2015-01-01

Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

Novel Biomarker Discovery for Diagnostic and Therapeutic Strategies in Prostate Cancer

DTIC Science & Technology

2015-06-01

PURPOSE: to identify high affinity aptamers that distinguish between prostate cancers that are likely to remain organ- confined and those with potential to...metastasize. SCOPE: This was a pilot project to generate RNA aptamers that selectively react with a prostate cancer cell line that remains confined... Aptamer -Facilitated Biomarker Discovery (AptaBiD) technology. TASKS AND PROGRESS: (1) Non-metastatic LNCaP-Pro-5 cells, metastasis-prone LNCaP-LN3

Direct electron-transfer conduits constructed at the interface between multicopper oxidase and nanocrystalline semiconductive Fe oxides

NASA Astrophysics Data System (ADS)

Nakamura, Ryuhei; Kamiya, Kazuhide; Hashimoto, Kazuhito

2010-10-01

Herein, the electron-transfer reactions occurring at the interface between bilirubin oxidase (BOD) and nanocrystalline hematite (α-Fe 2O 3) were characterized. Cyclic voltammograms indicated that BOD has an affinity for hematite surfaces and establishes a direct electron-transfer (DET) conduit between the primary electron acceptor T1 site and the conduction band of α-Fe 2O 3. DET was also confirmed photo-electrochemically, as cathodic photocurrents were generated when a nanocomposite of BOD and α-Fe 2O 3 was illuminated under oxygenated conditions. A proline residue displayed a high-binding affinity for hematite surfaces and is therefore likely part of an orientation-controlled motif which serves to locate BOD at the T1 site at a suitable distance for DET to α-Fe 2O 3.

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

PubMed Central

Sherwood, Laura J.; Hayhurst, Andrew

2013-01-01

Background Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. Methods and Findings In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. Conclusions The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus. PMID:23577211

First Penicillin-Binding Protein Occupancy Patterns of β-Lactams and β-Lactamase Inhibitors in Klebsiella pneumoniae.

PubMed

Sutaria, Dhruvitkumar S; Moya, Bartolome; Green, Kari B; Kim, Tae Hwan; Tao, Xun; Jiao, Yuanyuan; Louie, Arnold; Drusano, George L; Bulitta, Jürgen B

2018-06-01

Penicillin-binding proteins (PBPs) are the high-affinity target sites of all β-lactam antibiotics in bacteria. It is well known that each β-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite β-lactams serving as the cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae , PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae PBP binding was determined using isolated membrane fractions from K. pneumoniae strains ATCC 43816 and ATCC 13883. Binding reactions were conducted using β-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After β-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC 50 ]) were reported as the β-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by β-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC 50 , <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC 50 , <0.0075 mg/liter) and avibactam (IC 50 , 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC 50 , 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC 50 , 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC 50 , 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae enable, for the first time, the rational design and optimization of double β-lactam and β-lactam-β-lactamase inhibitor combinations. Copyright © 2018 American Society for Microbiology.

Hierarchy and Assortativity as New Tools for Binding-Affinity Investigation: The Case of the TBA Aptamer-Ligand Complex.

PubMed

Cataldo, Rosella; Alfinito, Eleonora; Reggiani, Lino

2017-12-01

Aptamers are single stranded DNA, RNA, or peptide sequences having the ability to bind several specific targets (proteins, molecules as well as ions). Therefore, aptamer production and selection for therapeutic and diagnostic applications is very challenging. Usually, they are generated in vitro, although computational approaches have been recently developed for the in silico production. Despite these efforts, the mechanism of aptamer-ligand formation is not completely clear, and producing high-affinity aptamers is still quite difficult. This paper aims to develop a computational model able to describe aptamer-ligand affinity. Topological tools, such as the conventional degree distribution, the rank-degree distribution (hierarchy), and the node assortativity are employed. In doing so, the macromolecules tertiary-structures are mapped into appropriate graphs. These graphs reproduce the main topological features of the macromolecules, by preserving the distances between amino acids (nucleotides). Calculations are applied to the thrombin binding aptamer (TBA), and the TBA-thrombin complex produced in the presence of Na + or K + . The topological analysis is able to detect several differences between complexes obtained in the presence of the two cations, as expected by previous investigations. These results support graph analysis as a novel computational tool for testing affinity. Otherwise, starting from the graphs, an electrical network can be obtained by using the specific electrical properties of amino acids and nucleobases. Therefore, a further analysis concerns with the electrical response, revealing that the resistance is sensitively affected by the presence of sodium or potassium, thus suggesting resistance as a useful physical parameter for testing binding affinity.

HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

PubMed Central

Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

2010-01-01

Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

PubMed Central

Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T.; Henry Dunand, Carole J.; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L.; Munroe, Melissa E.; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A.; Wilson, Patrick C.

2015-01-01

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus. PMID:25951191

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity.

PubMed

Chanda, Nripen; Kattumuri, Vijaya; Shukla, Ravi; Zambre, Ajit; Katti, Kavita; Upendran, Anandhi; Kulkarni, Rajesh R; Kan, Para; Fent, Genevieve M; Casteel, Stan W; Smith, C Jeffrey; Boote, Evan; Robertson, J David; Cutler, Cathy; Lever, John R; Katti, Kattesh V; Kannan, Raghuraman

2010-05-11

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC(50)) of AuNP-BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP-BBN and its radiolabeled surrogate (198)AuNP-BBN, exhibiting high binding affinity (IC(50) in microgram ranges), provide unequivocal evidence that AuNP-BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP-BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP-BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).

Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

PubMed

Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

2000-08-01

The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

[Paroxysmal perceptual alteration in comparison with hallucination--a review of its clinical reports and discussion of its pathophysiological mechanism in the present day, when second generation antipsychotics are widely used].

PubMed

Watanabe, Ken

2009-01-01

The syndrome of paroxysmal perceptual alteration (PPA) was first described by Yamaguchi in 1985. Since then, many PPA cases have been reported, and its pathophysiological mechanism has been proposed: a suppressed (blocked) mesolimbic and mesocortical dopaminergic system and sequential compensatory increase of noradrenergic neuronal activity are crucial for the occurrence of PPA. PPA is characterized by hypersensitivity of perception, psychedelic experience (brightening of colors, sharpening of contrast, visual distortion, etc.), and somatic schema disorder (one feels that one is floating, one's extremities are being pulled and elongated, etc.). PPA in chronic schizophrenic patients occurs abruptly like an attack mainly in the evening, often precipitated by fatigue. During the attack, patients also suffer from mood and thought alteration (anxiety, agitation, depressive mood, and inability to distract their thoughts from one thing), but they are aware that symptoms of PPA are not real and apprehensive about them. The attack ceases gradually and spontaneously while the patient rests or sleeps. These clinical features are clearly different from those of schizophrenic hallucinations. It is believed that PPA is closely related to neuroleptic treatment by conventional antipsychotics. I reported the prevalence of PPA as 4.0% in 1991 when high potential D2 blocking agents were prevailing. The occurrence of PPA has been significantly reduced to the present, when second generation (atypical) antipsychotics are prevailing. However, in my inquiry in 2004, the prevalence of PPA was 3.6% in cases treated with risperidone (RIS), while the rates were 0 in cases treated with olanzapine (OLZ), quetiapine (QTP), and perospirone (PRS). Several cases of PPA have been reported in patients who were treated with OLZ and PRS. Until now, no cases of PPA have been reported who were treated with QTP and aripiprazole (APZ). The prevalence of PPA among cases treated with these second generation antipsychotics might be related to the differences in these agents regarding their affinity for the D2 receptor: RIS has a sustained and close binding affinity, which might be similar to those of conventional antipsychotics, OLZ shows a sustained and loose binding affinity, PRS exhibits a transient and close binding affinity, whereas QTP has a transient and loose binding affinity. APZ is a partial agonist of the D2 receptor; APZ acts as an agonist under the condition of intrinsic dopaminergic dysfunction, which might prevent the occurrence of PPA.

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin*♦

PubMed Central

Samuel, Premila P.; Smith, Lucian P.; Phillips, George N.; Olson, John S.

2015-01-01

Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements. PMID:26205820

Development of a New De Novo Design Algorithm for Exploring Chemical Space.

PubMed

Mishima, Kazuaki; Kaneko, Hiromasa; Funatsu, Kimito

2014-12-01

In the first stage of development of new drugs, various lead compounds with high activity are required. To design such compounds, we focus on chemical space defined by structural descriptors. New compounds close to areas where highly active compounds exist will show the same degree of activity. We have developed a new de novo design system to search a target area in chemical space. First, highly active compounds are manually selected as initial seeds. Then, the seeds are entered into our system, and structures slightly different from the seeds are generated and pooled. Next, seeds are selected from the new structure pool based on the distance from target coordinates on the map. To test the algorithm, we used two datasets of ligand binding affinity and showed that the proposed generator could produce diverse virtual compounds that had high activity in docking simulations. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man*

PubMed Central

Macauley, Matthew S.; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M.; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C.

2015-01-01

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. PMID:26507663

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man.

PubMed

Macauley, Matthew S; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C

2015-12-11

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential effects of ethanol and other inducers of drug metabolism on the two forms of hamster liver microsomal aniline hydroxylase.

PubMed

McCoy, G D

1980-03-01

The aniline hydroxylase activity of microsomes isolated from hamster liver can be differentiated kinetically into high affinity (low K(m), form I) and low affinity (high K(m), form II) forms. Microsomes isolated from uninduced animals contain slightly more form I activity. The activity of the low affinity form (form II) is preferentially enhanced by Aroclor or 3-methylcholanthrene treatment, while phenobarbital treatment increases the activity of both forms. Chronic ethanol consumption results in enhancement of only the high affinity form (form I).

THE GENERALIZATION OF SIERPINSKI CARPET AND MENGER SPONGE IN n-DIMENSIONAL SPACE

NASA Astrophysics Data System (ADS)

Yang, Yun; Feng, Yuting; Yu, Yanhua

In this paper, we generalize Sierpinski carpet and Menger sponge in n-dimensional space, by using the generations and characterizations of affinely-equivalent Sierpinski carpet and Menger sponge. Exactly, Menger sponge in 4-dimensional space could be drawn out clearly under an affine transformation. Furthermore, the method could be used to a much broader class in fractals.

Purine uptake in Plasmodium: transport versus metabolism.

PubMed

Kirk, Kiaran; Howitt, Susan M; Bröer, Stefan; Saliba, Kevin J; Downie, Megan J

2009-06-01

In a recent paper, Quashie et al. have proposed that purine uptake into the intraerythrocytic malaria parasite involves four different plasma membrane transporters - two high affinity and two low affinity. They equate one of the two high-affinity transporters with PfNT1, a transporter reported previously to be a low-affinity system. Here, we offer an alternative interpretation of their data, suggesting that the conclusions drawn by Quashie et al. take insufficient account of metabolism.

Host-Guest Complexes with Protein-Ligand-Like Affinities: Computational Analysis and Design

PubMed Central

Moghaddam, Sarvin; Inoue, Yoshihisa

2009-01-01

It has recently been discovered that guests combining a nonpolar core with cationic substituents bind cucurbit[7]uril (CB[7]) in water with ultra-high affinities. The present study uses the Mining Minima algorithm to study the physics of these extraordinary associations and to computationally test a new series of CB[7] ligands designed to bind with similarly high affinity. The calculations reproduce key experimental observations regarding the affinities of ferrocene-based guests with CB[7] and β-cyclodextrin and provide a coherent view of the roles of electrostatics and configurational entropy as determinants of affinity in these systems. The newly designed series of compounds is based on a bicyclo[2.2.2]octane core, which is similar in size and polarity to the ferrocene core of the existing series. Mining Minima predicts that these new compounds will, like the ferrocenes, bind CB[7] with extremely high affinities. PMID:19133781

GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies

PubMed Central

Moelleken, Jörg; Gassner, Christian; Lingke, Sabine; Tomaschek, Simone; Tyshchuk, Oksana; Lorenz, Stefan; Mølhøj, Michael

2017-01-01

ABSTRACT The determination of the binding strength of immunoglobulins (IgGs) to targets can be influenced by avidity when the targets are soluble di- or multimeric proteins, or associated to cell surfaces, including surfaces introduced from heterogeneous assays. However, for the understanding of the contribution of a second drug-to-target binding site in molecular design, or for ranking of monovalent binders during lead identification, affinity-based assessment of the binding strength is required. Typically, monovalent binders like antigen-binding fragments (Fabs) are generated by proteolytic cleavage with papain, which often results in a combination of under- and over-digestion, and requires specific optimization and chromatographic purification of the desired Fabs. Alternatively, the Fabs are produced by recombinant approaches. Here, we report a lean approach for the functional assessment of human IgG1s during lead identification based on an in-solution digestion with the GingisKHAN™ protease, generating a homogenous pool of intact Fabs and Fcs and enabling direct assaying of the Fab in the digestion mixture. The digest with GingisKHAN™ is highly specific and quantitative, does not require much optimization, and the protease does not interfere with methods typically applied for lead identification, such as surface plasmon resonance or cell-based assays. GingisKHAN™ is highly suited to differentiate between affinity and avidity driven binding of human IgG1 monoclonal and bispecific antibodies during lead identification. PMID:28805498

High-affinity K+ uptake in pepper plants.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2005-06-01

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

T regulatory cells participate in the control of germinal centre reactions

PubMed Central

Alexander, Carla-Maria; Tygrett, Lorraine T; Boyden, Alexander W; Wolniak, Kristy L; Legge, Kevin L; Waldschmidt, Thomas J

2011-01-01

Germinal centre (GC) reactions are central features of T-cell-driven B-cell responses, and the site where antibody-producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity antibodies. These processes require exquisite regulation not only to ensure the production of desired antibodies, but to minimize unwanted autoreactive or low affinity antibodies. To assess whether T regulatory (Treg) cells participate in the control of GC responses, immunized mice were treated with an anti-glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) monoclonal antibody (mAb) to disrupt Treg-cell activity. In anti-GITR-treated mice, the GC B-cell pool was significantly larger compared with control-treated animals, with switched GC B cells composing an abnormally high proportion of the response. Dysregulated GCs were also observed regardless of strain, T helper type 1 or 2 polarizing antigens, and were also seen after anti-CD25 mAb treatment. Within the spleens of immunized mice, CXCR5+ and CCR7− Treg cells were documented by flow cytometry and Foxp3+ cells were found within GCs using immunohistology. Final studies demonstrated administration of either anti-transforming growth factor-β or anti-interleukin-10 receptor blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Treg cells is important in controlling the GC response. Taken together, these findings indicate that Treg cells contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs. PMID:21635248

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

PubMed

Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul

2016-02-01

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Pseudomonas aeruginosa reveals high intrinsic resistance to penem antibiotics: penem resistance mechanisms and their interplay.

PubMed

Okamoto, K; Gotoh, N; Nishino, T

2001-07-01

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC beta-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC beta-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC beta-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC beta-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC beta-lactamase but not from the lower affinity of PBPs for penems.

Pseudomonas aeruginosa Reveals High Intrinsic Resistance to Penem Antibiotics: Penem Resistance Mechanisms and Their Interplay

PubMed Central

Okamoto, Kiyomi; Gotoh, Naomasa; Nishino, Takeshi

2001-01-01

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC β-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC β-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC β-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC β-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC β-lactamase but not from the lower affinity of PBPs for penems. PMID:11408209

Pertussis toxin modifies the characteristics of both the inhibitory GTP binding proteins and the somatostatin receptor in anterior pituitary tumor cells

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

Mahy, N.; Woolkalis, M.; Thermos, K.

1988-08-01

The effects of pertussis toxin treatment on the characteristics of somatostatin receptors in the anterior pituitary tumor cell line AtT-20 were examined. Pertussis toxin selectively catalyzed the ADP ribosylation of the alpha subunits of the inhibitory GTP binding proteins in AtT-20 cells. Toxin treatment abolished somatostatin inhibition of forskolin-stimulated adenylyl cyclase activity and somatostatin stimulation of GTPase activity. To examine the effects of pertussis toxin treatment on the characteristics of the somatostatin receptor, the receptor was labeled by the somatostatin analog (125I)CGP 23996. (125I)CGP 23996 binding to AtT-20 cell membranes was saturable and within a limited concentration range was tomore » a single high affinity site. Pertussis toxin treatment reduced the apparent density of the high affinity (125I)CGP 23996 binding sites in AtT-20 cell membranes. Inhibition of (125I)CGP 23996 binding by a wide concentration range of CGP 23996 revealed the presence of two binding sites. GTP predominantly reduced the level of high affinity sites in control membranes. Pertussis toxin treatment also diminished the amount of high affinity sites. GTP did not affect (125I)CGP 23996 binding in the pertussis toxin-treated membranes. The high affinity somatostatin receptors were covalently labeled with (125I) CGP 23996 and the photoactivated crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. No high affinity somatostatin receptors, covalently bound to (125I)CGP 23996, were detected in the pertussis toxin-treated membranes. These results are most consistent with pertussis toxin uncoupling the inhibitory G proteins from the somatostatin receptor thereby converting the receptor from a mixed population of high and low affinity sites to only low affinity receptors.« less

Kinetics of N-Utilization By Natural Phytoplankton Assemblages During Upwelling Events At The NW Iberian Shelf

NASA Astrophysics Data System (ADS)

Brion, N.; Elskens, M.; Dehairs, F.; Baeyens, W.

2003-04-01

The concentration-dependent uptakes of nitrate, ammonium and the effect of ammo-nium on the f-ratio were surveyed in surface waters of the NW Iberian shelf during June 1997, July 1998 and September 1999. Because relationships between rates and substrate concentrations were quite variable, ranging from linear to convex shaped curves, they were fitted to rational functions. Stepwize regression analysis yielded subsequent model equations with reasonable statistical properties which allowed describing all but all a few cases. Differentiating these equations with respect to the concentration gave the slope of the tangent to the curve, i.e., the variation in rate expected for a given perturbation of the ambient substrate concentration. The initial slope value was then used as an index to gauge the "affinity" of the plankton community for the nitrogen substrate utilization. In June 1997, the situation at the Iberian shelf showed no upwelling except near Cape Finistère. Overall, the phytoplankton community displayed a high "affinity" for both nitrate and ammonium and low f-ratio values, which is indicative of a re-generated production regime. High ammonium regeneration rates supported furthermore these observations. It was also demonstrated that the new production rates is only marginally sensitive to changes of the ambient nitrate and/or ammonium concentrations. This indicates that the production regime is rather stable throughout. Only at Cape Finistère, nitrate concentrations were high reflecting the onset of an upwelling event. In this zone, the phytoplankton community, taking advantage of its high affinity for nitrate enhanced both total N-uptake rate and f-ratio. In July 1998, the situation evolved towards an extension to the south of the upwelling event starting at Cape Finistère. In this southern zone of the upwelling the phytoplankton community displayed generally a lower affinity for nitrate (but not for ammonium) than in 1997. In spite of this lower affinity, nitrate uptake rate was dominant resulting in f-ratio values greater than 0.5, a characteristic of a new production regime. The new production rate is only marginally sensitive to increases of the ambient nitrate, but is drastically inhibited by small increases of the ambient ammonium. The situation of September 1999 was very close to that observed in July 1998, with higher nitrate concentrations in the coastal northern part of the sampling area dominated by upwelling.

Cytomegalovirus-Specific CD8+ T-Cells With Different T-Cell Receptor Affinities Segregate T-Cell Phenotypes and Correlate With Chronic Graft-Versus-Host Disease in Patients Post-Hematopoietic Stem Cell Transplantation

PubMed Central

Poiret, Thomas; Axelsson-Robertson, Rebecca; Remberger, Mats; Luo, Xiao-Hua; Rao, Martin; Nagchowdhury, Anurupa; Von Landenberg, Anna; Ernberg, Ingemar; Ringden, Olle; Maeurer, Markus

2018-01-01

Virus-specific T-cell responses are crucial to control cytomegalovirus (CMV) infections/reactivation in immunocompromised individuals. Adoptive cellular therapy with CMV-specific T-cells has become a viable treatment option. High-affinity anti-viral cellular immune responses are associated with improved long-term immune protection against CMV infection. To date, the characterization of high-affinity T-cell responses against CMV has not been achieved in blood from patients after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, the purpose of this study was to describe and analyze the phenotype and clinical impact of different CMV-specific CD8+ cytotoxic T-lymphocytes (CMV-CTL) classes based on their T-cell receptor (TCR) affinity. T-cells isolated from 23 patients during the first year following HSCT were tested for the expression of memory markers, programmed cell death 1 (PD-1), as well as TCR affinity, using three different HLA-A*02:01 CMVNLVPMVATV-Pp65 tetramers (wild-type, a245v and q226a mutants). High-affinity CMV-CTL defined by q226a tetramer binding, exhibited a higher frequency in CD8+ T-cells in the first month post-HSCT and exhibited an effector memory phenotype associated with strong PD-1 expression as compared to the medium- and low-affinity CMV-CTLs. High-affinity CMV-CTL was found at higher proportion in patients with chronic graft-versus-host disease (p < 0.001). This study provides a first insight into the detailed TCR affinities of CMV-CTL. This may be useful in order to improve current immunotherapy protocols using isolation of viral-specific T-cell populations based on their TCR affinity. PMID:29692783

Molecular mechanism of immunoglobulin V-region diversification regulated by transcription and RNA metabolism in antigen-driven B cells.

PubMed

Sakaguchi, N; Maeda, K; Kuwahara, K

2011-06-01

The immune system produces specific antibodies (Ab) against any antigens (Ag) of exogenous and endogenous origins with a diverse repertoire of V-region specificities. The primary V-region repertoire is created by the rearrangement of immunoglobulin (Ig) V-region, D- and J-segments with the insertion of N- and P-sequences during early B cell differentiation. Recent studies revealed that secondary diversification of the IgV-region generated in the peripheral lymphoid organs plays a critical role in the generation of effective Ab production for protection from various pathogens. Naïve B cells that react with Ags initiate proliferation and differentiation in the follicular region and create the germinal centres (GCs), where activation-induced cytidine deaminase (AID)-dependent IgV-region somatic hypermutation (SHM) and class-switch recombination generate high-affinity and class-switched mature Ag-specific B cells. Our studies have discovered a 210-kDa nuclear protein, named GC-associated nuclear protein (GANP) that is up-regulated in GC B cells during the T cell-dependent (TD) immune responses. By studying mice with mutant forms of the ganp gene, we demonstrated that GANP is essential for the generation of high-affinity B cells against TD-Ag by affecting SHM at the IgV-regions. GANP is associated with AID in the cytoplasm and the GANP/AID complex is recruited to the nucleus, specifically, the chromatin, and targeted selectively to the IgV-region gene in B cells. GANP augments the access of AID towards IgV-regions in B cells. Here, we review the role of GANP in acquired immunity through the detailed analysis of the molecular mechanism generating SHM specifically at IgV-regions in B cells. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion

PubMed Central

Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario

2016-01-01

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity–primed T cells acquired cytotoxic activity earlier than high affinity–primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity–stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. PMID:27799622

Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics.

PubMed

Seeman, P; Ko, F; Tallerico, T

2005-09-01

Although phencyclidine and ketamine are used to model a hypoglutamate theory of schizophrenia, their selectivity for NMDA receptors has been questioned. To determine the affinities of phencyclidine, ketamine, dizocilpine and LSD for the functional high-affinity state of the dopamine D2 receptor, D2High, their dissociation constants (Ki) were obtained on [3H]domperidone binding to human cloned dopamine D2 receptors. Phencyclidine had a high affinity for D2High with a Ki of 2.7 nM, in contrast to its low affinity for the NMDA receptor, with a Ki of 313 nM, as labeled by [3H]dizocilpine on rat striatal tissue. Ketamine also had a high affinity for D2High with a Ki of 55 nM, an affinity higher than its 3100 nM Ki for the NMDA sites. Dizocilpine had a Ki of 0.3 nM at D2High, but a Kd of 1.8 nM at the NMDA receptor. LSD had a Ki of 2 nM at D2High. Because the psychotomimetics had higher potency at D2High than at the NMDA site, the psychotomimetic action of these drugs must have a major contribution from D2 agonism. Because these drugs have a combined action on both dopamine receptors and NMDA receptors, these drugs, when given in vivo, test a combined hyperdopamine and hypoglutamate theory of psychosis.

Boosting Affinity by Correct Ligand Preorganization for the S2 Pocket of Thrombin: A Study by Isothermal Titration Calorimetry, Molecular Dynamics, and High-Resolution Crystal Structures.

PubMed

Rühmann, Eggert H; Rupp, Melinda; Betz, Michael; Heine, Andreas; Klebe, Gerhard

2016-02-04

Structural preorganization to fix bioactive conformations at protein binding sites is a popular strategy to enhance binding affinity during late-stage optimization. The rationale for this enhancement relates to entropic advantages assigned to rigidified versus flexible ligands. We analyzed a narrow series of peptidomimetics binding to thrombin. The individual ligands exhibit at P2 a conformationally flexible glycine, more restricted alanine, N-methylglycine, N-methylhomoalanine, and largely rigidified proline moiety. Overall, affinity was found to increase by a factor of 1000, explained partly by an entropic advantage. All ligands adopt the same binding mode with small deviations. The residual mobility of the bound ligands is decreased across the series, and a protein side chain differs in its order/disorder behavior along with changes in the surface-water network pattern established across the newly generated protein-ligand surfaces. The enthalpy/entropy inventory displays a rather complex picture and emphasizes that thermodynamics can only be compared in terms of relative differences within a structurally similar ligand series. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Potential toxicity and affinity of triphenylmethane dye malachite green to lysozyme.

PubMed

Ding, Fei; Li, Xiu-Nan; Diao, Jian-Xiong; Sun, Ye; Zhang, Li; Ma, Lin; Yang, Xin-Ling; Zhang, Li; Sun, Ying

2012-04-01

Malachite green is a triphenylmethane dye that is used extensively in many industrial and aquacultural processes, generating environmental concerns and health problems to human being. In this contribution, the complexation between lysozyme and malachite green was verified by means of computer-aided molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) approaches. The precise binding patch of malachite green in lysozyme has been identified from molecular modeling and ANS displacement, Trp-62, Trp-63, and Trp-108 residues of lysozyme were earmarked to possess high-affinity for this dye, the principal forces in the lysozyme-malachite green adduct are hydrophobic and π-π interactions. Steady state fluorescence proclaimed the complex of malachite green with lysozyme yields quenching through static type, which substantiates time-resolved fluorescence measurements that lysozyme-malachite green conjugation formation has an affinity of 10(3)M(-1). Moreover, via molecular modeling and also CD data, we can safely arrive at a conclusion that the polypeptide chain of lysozyme partially destabilized upon complexation with malachite green. The data emerged here will help to further understand the toxicological action of malachite green in human body. Copyright © 2012 Elsevier Inc. All rights reserved.

Fiber optic evanescent wave biosensor

NASA Astrophysics Data System (ADS)

Duveneck, Gert L.; Ehrat, Markus; Widmer, H. M.

1991-09-01

The role of modern analytical chemistry is not restricted to quality control and environmental surveillance, but has been extended to process control using on-line analytical techniques. Besides industrial applications, highly specific, ultra-sensitive biochemical analysis becomes increasingly important as a diagnostic tool, both in central clinical laboratories and in the doctor's office. Fiber optic sensor technology can fulfill many of the requirements for both types of applications. As an example, the experimental arrangement of a fiber optic sensor for biochemical affinity assays is presented. The evanescent electromagnetic field, associated with a light ray guided in an optical fiber, is used for the excitation of luminescence labels attached to the biomolecules in solution to be analyzed. Due to the small penetration depth of the evanescent field into the medium, the generation of luminescence is restricted to the close proximity of the fiber, where, e.g., the luminescent analyte molecules combine with their affinity partners, which are immobilized on the fiber. Both cw- and pulsed light excitation can be used in evanescent wave sensor technology, enabling the on-line observation of an affinity assay on a macroscopic time scale (seconds and minutes), as well as on a microscopic, molecular time scale (nanoseconds or microseconds).

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma.

PubMed

Ikonomopoulou, M P; Bradley, A J; Whittier, J M; Ibrahim, K

2006-11-01

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4 degrees C with high affinity (K (a) = 1.49 +/- 0.09 x 10(9) M(-1); 0.17 +/- 0.02 x 10(7) M(-1)) and low binding capacity (B (max) = 3.24 +/- 0.84 x 10(-5) M; 0.33 +/- 0.06 x 10(-4) M). The binding affinity and capacity of testosterone at 23 and 36 degrees C, respectively were similar to those determined at 4 degrees C. However, oestradiol showed no binding activity at 36 degrees C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36 degrees C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.

Extra Double-stranded RNA Binding Domain (dsRBD) in a Squid RNA Editing Enzyme Confers Resistance to High Salt Environment*

PubMed Central

Palavicini, Juan Pablo; Correa-Rojas, Rodrigo A.; Rosenthal, Joshua J. C.

2012-01-01

A-to-I RNA editing is particularly common in coding regions of squid mRNAs. Previously, we isolated a squid editing enzyme (sqADAR2) that shows a unique structural feature when compared with other ADAR2 family members: an additional double-stranded RNA (dsRNA) binding domain (dsRBD). Alternative splicing includes or excludes this motif, generating a novel or a conventional variant termed sqADAR2a and sqADAR2b, respectively. The extra dsRBD of sqADAR2a increases its editing activity in vitro. We hypothesized that the high activity is due to an increase in the affinity of the enzyme for dsRNA. This may be important because protein-RNA interactions can be influenced by physical factors. We became particularly interested in analyzing the effects of salt on interactions between sqADAR2 and RNA because squid cells have a ∼3-fold higher ionic strength and proportionally more Cl− than vertebrate cells. To date, in vitro biochemical analyses of adenosine deamination have been conducted using vertebrate-like ionic strength buffers containing chloride as the major anion, although the vast majority of cellular anions are known to be organic. We found that squid-like salt conditions severely impair the binding affinity of conventional ADAR2s for dsRNA, leading to a decrease in nonspecific and site-specific editing activity. Inhibition of editing was mostly due to high Cl− levels and not to the high concentrations of K+, Na+, and organic anions like glutamate. Interestingly, the extra dsRBD in sqADAR2a conferred resistance to the high Cl− levels found in squid neurons. It does so by increasing the affinity of sqADAR2 for dsRNA by 30- or 100-fold in vertebrate-like or squid-like conditions, respectively. Site-directed mutagenesis of squid ADAR2a showed that its increased affinity and editing activity are directly attributable to the RNA binding activity of the extra dsRBD. PMID:22457361

Immobilization of CdS nanoparticles formed in reverse micelles onto aluminosilicate supports and their photocatalytic properties.

PubMed

Hirai, Takayuki; Bando, Yoko

2005-08-15

CdS nanoparticles, prepared in reverse micellar system, were immobilized onto thiol-modified aluminosilicate particles (ASSH) by a simple operation: addition of ASSH in the micellar solution and mild stirring. The resulting CdS nanoparticles-aluminosilicate composites (ASCdS) were used as photocatalysts for H2 generation from 2-propanol aqueous solution. The chemical properties of the aluminosilicate, such as affinity for water and other reactants, were found to affect the photocatalytic property of the CdS nanoparticles immobilized. Zeolite particles, having affinity for water and 2-propanol, gave a good ASCdS photocatalyst with respect to H2 generation.

Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells

PubMed Central

1993-01-01

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD- deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell- independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen- driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens. PMID:8418208

Evidence that human immunoglobulin M rheumatoid factors can Be derived from the natural autoantibody pool and undergo an antigen driven immune response in which somatically mutated rheumatoid factors have lower affinities for immunoglobulin G Fc than their germline counterparts.

PubMed

Carayannopoulos, M O; Potter, K N; Li, Y; Natvig, J B; Capra, J D

2000-04-01

The question of whether immunoglobulin (Ig)M rheumatoid factors (RF) arise as the result of an abnormal expansion of already existing clones producing natural autoantibodies or emerge as new clones that are somatically mutated owing to an antigen driven immune response has never been conclusively answered. In this study, an inhibition ELISA was utilized to measure the affinities of recombinant antibodies using VH segments reverted back to their closest germline counterparts (germline revertants). In all cases, the somatically mutated parental RFs had a decreased affinity for immunoglobulin (Ig)G Fc compared to the germline revertant, indicating that the antibodies in the germline configuration had the higher affinities. This demonstrates that somatic mutation is not a prerequisite to generate disease associated antibodies. The presence of mutations in the parental IgM RFS suggests that these cells had been involved in a germinal centre reaction. As the germinal centre is the conventional site of the acquisition of mutations during an antigen driven response, these data suggest a role for germinal centres in the generation of the antibody diversity in addition to the selection of higher affinity antibodies. Assuming that only antigen selected cells survive deletion, these data support the hypothesis that IgM RFS can be derived from the natural autoantibody repertoire and result from an antigen driven response. Mechanisms controlling the survival of B cells based on the affinity/avidity of the immunoglobulin receptor are shown to be functional in patients with rheumatoid arthritis.

The competitive advantage of a dual-transporter system.

PubMed

Levy, Sagi; Kafri, Moshe; Carmi, Miri; Barkai, Naama

2011-12-09

Cells use transporters of different affinities to regulate nutrient influx. When nutrients are depleted, low-affinity transporters are replaced by high-affinity ones. High-affinity transporters are helpful when concentrations of nutrients are low, but the advantage of reducing their abundance when nutrients are abundant is less clear. When we eliminated such reduced production of the Saccharomyces cerevisiae high-affinity transporters for phosphate and zinc, the elapsed time from the initiation of the starvation program until the lack of nutrients limited growth was shortened, and recovery from starvation was delayed. The latter phenotype was rescued by constitutive activation of the starvation program. Dual-transporter systems appear to prolong preparation for starvation and to facilitate subsequent recovery, which may optimize sensing of nutrient depletion by integrating internal and external information about nutrient availability.

A structure-function study of PACAP using conformationally-restricted analogs: identification of PAC1 receptor-selective PACAP agonists

PubMed Central

Ramos-Álvarez, Irene; Mantey, Samuel A.; Nakamura, Taichi; Nuche-Berenguer, Bernardo; Moreno, Paola; Moody, Terry W.; Maderdrut, Jerome L.; Coy, David H.; Jensen, Robert T.

2015-01-01

Pituitary adenylate-cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure-function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally-restricted PACAP -analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22 ,28,34,38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to-103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases. PMID:25698233

A HIGH-LEVEL CALCULATION OF THE PROTON AFFINITY OF DIBORANE

EPA Science Inventory

The experimental proton affinity of diborane (B2H6) is based on an unstable species, B2H,+, 4 which has been observed only at low temperatures. The present work calculates the proton 5 affinity of diborane using the Gaussian-3 method and other high-level compound ab initio 6 met...

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

PubMed

Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

2016-10-01

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

Synthesis and biological evaluation of new 2-(4,5-dihydro-1H-imidazol-2-yl)-3,4-dihydro-2H-1,4-benzoxazine derivatives.

PubMed

Touzeau, Frédérique; Arrault, Axelle; Guillaumet, Gérald; Scalbert, Elizabeth; Pfeiffer, Bruno; Rettori, Marie-Claire; Renard, Pierre; Mérour, Jean-Yves

2003-05-08

2-(4,5-Dihydro-1H-imidazol-2-yl)-3,4-dihydro-2H-1,4-benzoxazine derivatives and tricyclic analogues with a fused additional ring on the nitrogen atom of the benzoxazine moiety have been prepared and evaluated for their cardiovascular effects as potential antihypertensive agents. The imidazoline ring was generated by reaction of the corresponding ethyl ester with ethylenediamine. Affinities for imidazoline binding sites (IBS) I(1) and I(2) and alpha(1) and alpha(2) adrenergic receptors were evaluated as well as the effects on mean arterial blood pressure (MAP) and heart rate (HR) of spontaneously hypertensive rats. With few exceptions the most active compounds on MAP were those with high affinities for IBS and alpha(2) receptor. Among these, compound 4h was the most interesting and is now, together with its enantiomers, under complementary pharmacological evaluation.

CO Dehydrogenase Genes Found in Metagenomic Fosmid Clones from the Deep Mediterranean Sea▿ †

PubMed Central

Martin-Cuadrado, Ana-Belen; Ghai, Rohit; Gonzaga, Aitor; Rodriguez-Valera, Francisco

2009-01-01

The use of carbon monoxide (CO) as a biological energy source is widespread in microbes. In recent years, the role of CO oxidation in superficial ocean waters has been shown to be an important energy supplement for heterotrophs (carboxydovores). The key enzyme CO dehydrogenase was found in both isolates and metagenomes from the ocean's photic zone, where CO is continuously generated by organic matter photolysis. We have also found genes that code for both forms I (low affinity) and II (high affinity) in fosmids from a metagenomic library generated from a 3,000-m depth in the Mediterranean Sea. Analysis of other metagenomic databases indicates that similar genes are also found in the mesopelagic and bathypelagic North Pacific and on the surfaces of this and other oceanic locations (in lower proportions and similarities). The frequency with which this gene was found indicates that this energy-generating metabolism would be at least as important in the bathypelagic habitat as it is in the photic zone. Although there are no data about CO concentrations or origins deep in the ocean, it could have a geothermal origin or be associated with anaerobic metabolism of organic matter. The identities of the microbes that carry out these processes were not established, but they seem to be representatives of either Bacteroidetes or Chloroflexi. PMID:19801465

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

PubMed

Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

2017-06-01

Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Characterization of two ammonia-oxidizing bacteria isolated from reactors operated with low dissolved oxygen concentrations.

PubMed

Park, H-D; Noguera, D R

2007-05-01

To obtain ammonia-oxidizing bacterial (AOB) strains inhabiting low dissolved oxygen (DO) environments and to characterize them to better understand their function and ecology. Using a serial dilution method, two AOB strains (ML1 and NL7) were isolated from chemostat reactors operated with low DO concentrations (0.12-0.24 mg l(-1)). Phylogenetically, strains ML1 and NL7 are affiliated to AOB within the Nitrosomonas europaea and Nitrosomonas oligotropha lineages, respectively. Kinetically, strain ML1 had high affinity for oxygen (0.24 +/- 0.13 mg l(-1)) and low affinity for ammonia (1.62 +/- 0.97 mg N l(-1)), while strain NL7 had high affinity for ammonia (0.48 +/- 0.35 mg l(-1)), but a surprisingly low affinity for oxygen (1.22 +/- 0.43 mg l(-1)). A co-culture experiment was used to iteratively estimate decay constants for both strains. The results indicated that AOB without high affinity for oxygen may have other mechanisms to persist in low DO environments, with high affinity for ammonia being important. This study provides a method to determine AOB growth kinetic parameters without assuming or neglecting decay constant. And, this is the first report on oxygen affinity constant of a N. oligotropha strain.

Pharmacological and gene regulation properties point to the SlHAK5 K+ transporter as a system for high-affinity Cs+ uptake in tomato plants.

PubMed

Ródenas, Reyes; Nieves-Cordones, Manuel; Rivero, Rosa M; Martinez, Vicente; Rubio, Francisco

2018-04-01

Potassium (K + ) and cesium (Cs + ) are chemically similar but while K + is an essential nutrient, Cs + can be toxic for living organisms, plants included. Two different situations could lead to problems derived from the presence of Cs + in agricultural systems: (1) presence of Cs + at high concentrations that could produce toxic effects on plants, (2) presence of micromolar concentrations of radiocesium, which can be accumulated in the plant and affect animal and human health through the food chain. While K + uptake has been well described in tomato plants, information on molecular mechanisms involved in Cs + accumulation in this species is absent. Here, we show that in tomato plants, high concentrations of Cs + produce deficiency of K + but do not induce high-affinity K + uptake or the gene encoding the high-affinity K + transporter SlHAK5. At these concentrations, Cs + uptake takes place through a Ca 2+ -sensitive pathway, probably a non-selective cation channel. At micromolar concentrations, Cs + is accumulated by a high-affinity uptake system upregulated in K + -starved plants. This high-affinity Cs + uptake shares features with high-affinity K + uptake. It is sensitive to NH 4 + and insensitive to Ba 2+ and Ca 2+ and its presence parallels the pattern of SlHAK5 expression. Moreover, blockers of reactive oxygen species and ethylene action repress SlHAK5 and negatively regulate both high-affinity K + and Cs + uptake. Thus, we propose that SlHAK5 contributes to Cs + uptake from micromolar concentrations in tomato plants and can constitute a pathway for radiocesium transfer from contaminated areas to the food chain. © 2017 Scandinavian Plant Physiology Society.

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

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

PubMed

Venkataraman, Anand; Yang, Kun; Irizarry, Jose; Mackiewicz, Mark; Mita, Paolo; Kuang, Zheng; Xue, Lin; Ghosh, Devlina; Liu, Shuang; Ramos, Pedro; Hu, Shaohui; Bayron Kain, Diane; Keegan, Sarah; Saul, Richard; Colantonio, Simona; Zhang, Hongyan; Behn, Florencia Pauli; Song, Guang; Albino, Edisa; Asencio, Lillyann; Ramos, Leonardo; Lugo, Luvir; Morell, Gloriner; Rivera, Javier; Ruiz, Kimberly; Almodovar, Ruth; Nazario, Luis; Murphy, Keven; Vargas, Ivan; Rivera-Pacheco, Zully Ann; Rosa, Christian; Vargas, Moises; McDade, Jessica; Clark, Brian S; Yoo, Sooyeon; Khambadkone, Seva G; de Melo, Jimmy; Stevanovic, Milanka; Jiang, Lizhi; Li, Yana; Yap, Wendy Y; Jones, Brittany; Tandon, Atul; Campbell, Elliot; Montelione, Gaetano T; Anderson, Stephen; Myers, Richard M; Boeke, Jef D; Fenyö, David; Whiteley, Gordon; Bader, Joel S; Pino, Ignacio; Eichinger, Daniel J; Zhu, Heng; Blackshaw, Seth

2018-03-19

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.

Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts.

PubMed

Yañez, Fernando; Chianella, Iva; Piletsky, Sergey A; Concheiro, Angel; Alvarez-Lorenzo, Carmen

2010-02-05

This work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA.HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross-linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA.HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g(-1) (i.e., 170 mg g(-1)) under flow (1 mL min(-1)) of 0.2 mM sodium cholate solution. Copyright 2009 Elsevier B.V. All rights reserved.

Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

PubMed

Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

1993-03-10

Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components

PubMed Central

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, UA

2014-01-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and Impact of the Study Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. PMID:24935714

Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy.

PubMed

Shlamkovich, Tomer; Aharon, Lidan; Barton, William A; Papo, Niv

2017-05-16

In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, F; Rasaee, M J; Forouzandeh Moghadam, M; Allameh, A A; Sadroddiny, E

2004-06-01

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

PubMed Central

Kadonosono, Tetsuya; Yabe, Etsuri; Furuta, Tadaomi; Yamano, Akihiro; Tsubaki, Takuya; Sekine, Takuya; Kuchimaru, Takahiro; Sakurai, Minoru; Kizaka-Kondoh, Shinae

2014-01-01

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides. PMID:25084350

Structural Insights into Antibody Sequestering and Neutralizing of Na+ Channel α-Type Modulator from Old World Scorpion Venom

PubMed Central

Fabrichny, Igor P.; Mondielli, Grégoire; Conrod, Sandrine; Martin-Eauclaire, Marie-France; Bourne, Yves; Marchot, Pascale

2012-01-01

The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage-activated Na+ channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 Å-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 Å-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin-bound anti-AahII Fab and planar combining surface of the unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy. PMID:22371498

Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3*

PubMed Central

Roy, Adhiraj; Kim, Jeong-Ho

2014-01-01

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations. PMID:24451370

Bohr effect and temperature sensitivity of hemoglobins from highland and lowland deer mice.

PubMed

Jensen, Birgitte; Storz, Jay F; Fago, Angela

2016-05-01

An important means of physiological adaptation to environmental hypoxia is an increased oxygen (O2) affinity of the hemoglobin (Hb) that can help secure high O2 saturation of arterial blood. However, the trade-off associated with a high Hb-O2 affinity is that it can compromise O2 unloading in the systemic capillaries. High-altitude deer mice (Peromyscus maniculatus) have evolved an increased Hb-O2 affinity relative to lowland conspecifics, but it is not known whether they have also evolved compensatory mechanisms to facilitate O2 unloading to respiring tissues. Here we investigate the effects of pH (Bohr effect) and temperature on the O2-affinity of high- and low-altitude deer mouse Hb variants, as these properties can potentially facilitate O2 unloading to metabolizing tissues. Our experiments revealed that Bohr factors for the high- and low-altitude Hb variants are very similar in spite of the differences in O2-affinity. The Bohr factors of deer mouse Hbs are also comparable to those of other mammalian Hbs. In contrast, the high- and low-altitude variants of deer mouse Hb exhibited similarly low temperature sensitivities that were independent of red blood cell anionic cofactors, suggesting an appreciable endothermic allosteric transition upon oxygenation. In conclusion, high-altitude deer mice have evolved an adaptive increase in Hb-O2 affinity, but this is not associated with compensatory changes in sensitivity to changes in pH or temperature. Instead, it appears that the elevated Hb-O2 affinity in high-altitude deer mice is compensated by an associated increase in the tissue diffusion capacity of O2 (via increased muscle capillarization), which promotes O2 unloading. Copyright © 2016 Elsevier Inc. All rights reserved.

Preparation and In Vitro Photodynamic Activity of Glucosylated Zinc(II) Phthalocyanines as Underlying Targeting Photosensitizers.

PubMed

Liu, Jian-Yong; Wang, Chen; Zhu, Chun-Hui; Zhang, Zhi-Hong; Xue, Jin-Ping

2017-05-19

Two novel glucosylated zinc(ІІ) phthalocyanines 7a-7b, as well as the acetyl-protected counterparts 6a-6b, have been synthesized by the Cu(I)-catalyzed 1,3-dipolar cycloaddition between the propargylated phthalocyanine and azide-substituted glucoses. All of these phthalocyanines were characterized with various spectroscopic methods and studied for their photo-physical, photo-chemical, and photo-biological properties. With glucose as the targeting unit, phthalocyanines 7a-7b exhibit a specific affinity to MCF-7 breast cancer cells over human embryonic lung fibroblast (HELF) cells, showing higher cellular uptake. Upon illumination, both photosensitizers show high cytotoxicity with IC 50 as low as 0.032 µM toward MCF-7 cells, which are attributed to their high cellular uptake and low aggregation tendency in the biological media, promoting the generation of intracellular reactive oxygen species (ROS). Confocal laser fluorescence microscopic studies have also revealed that they have high and selective affinities to the lysosomes, but not the mitochondria, of MCF-7 cells. The results show that these two glucosylated zinc(II) phthalocyanines are potential anticancer agents for targeting photodynamic therapy.

Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis.

PubMed

Chen, Jia-Liang; Zhao, Yu; Gong, Yan-Jun; Pan, Bin-Bin; Wang, Xiao; Su, Xun-Cheng

2018-02-01

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange

DOE PAGES

Pennington, Luke F.; Tarchevskaya, Svetlana; Brigger, Daniel; ...

2016-05-19

Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab–Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, inmore » combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. Furthermore, this combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions.« less

Bisubstrate inhibitors of protein kinases: from principle to practical applications.

PubMed

Lavogina, Darja; Enkvist, Erki; Uri, Asko

2010-01-01

Bisubstrate inhibitors consist of two conjugated fragments, each targeted to a different binding site of a bisubstrate enzyme. The design of bisubstrate inhibitors presupposes the formation of the ternary complex in the course of the catalyzed reaction. The principle advantage of bisubstrate inhibitors is their ability to generate more interactions with the target enzyme that could result in improved affinity and selectivity of the conjugates, when compared with single-site inhibitors. Among phosphotransferases, the approach was first successfully used for adenylate kinase in 1973. Since then, several types of bisubstrate inhibitors have been developed for protein kinases, including conjugates of peptides with nucleotides, adenosine derivatives and potent ATP-competitive inhibitors. Earlier bisubstrate inhibitors had pharmacokinetic qualities that were unsuitable for cellular experiments and hence were mostly used for in vitro studies. The recently constructed conjugates of adenosine derivatives and D-arginine-rich peptides (ARCs) possess high kinase affinity, high biological and chemical stability and good cell plasma membrane penetrative properties that enable their application in the regulation of cellular protein phosphorylation balances in cell and tissue experiments.

Quantitative interaction proteomics using mass spectrometry.

PubMed

Wepf, Alexander; Glatter, Timo; Schmidt, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

2009-03-01

We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

Direct Measurement of Equilibrium Constants for High-Affinity Hemoglobins

PubMed Central

Kundu, Suman; Premer, Scott A.; Hoy, Julie A.; Trent, James T.; Hargrove, Mark S.

2003-01-01

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (KD ≪ 1 μM) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and ∼100 μM−1 respectively, indicate that they are not capable of facilitating oxygen transport. PMID:12770899

Direct Measurement of T Cell Receptor Affinity and Sequence from Naïve Anti-Viral T Cells

PubMed Central

Zhang, Shuqi; Parker, Patricia; Ma, Keyue; He, Chenfeng; Shi, Qian; Cui, Zhonghao; Williams, Chad; Wendel, Ben S.; Meriwether, Amanda; Salazar, Mary A.; Jiang, Ning

2016-01-01

T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCR). The affinity of TCR to cognate antigen is of high interest in adoptive T cell transfer immunotherapy and antigen-specific T cell repertoire immune profiling because it is widely known to correlate with downstream T cell responses. Here, we introduce the in situ TCR affinity and sequence test (iTAST) for simultaneous measurement of TCR affinity and sequence from single primary CD8+ T cells in human blood. We demonstrate that the repertoire of primary antigen-specific T cells from pathogen inexperienced individuals has a surprisingly broad affinity range of 1000-fold composed of diverse TCR sequences. Within this range, samples from older individuals contained a reduced frequency of high affinity T cells compared to young individuals, demonstrating an age-related effect of T cell attrition that could cause holes in the repertoire. iTAST should enable the rapid selection of high affinity TCRs ex vivo for adoptive immunotherapy and measurement of T cell response for immune monitoring applications. PMID:27252176

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Rani, Mridula; Bolles, Meagan; Donaldson, Eric F.; Van Blarcom, Thomas; Baric, Ralph; Iverson, Brent

2012-01-01

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection. PMID:22696652

Pushing antibody-based labeling systems to higher sensitivity by linker-assisted affinity enhancement.

PubMed

Gorris, Hans H; Bade, Steffen; Röckendorf, Niels; Fránek, Milan; Frey, Andreas

2011-08-17

The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

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

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Extension arm facilitated pegylation of alphaalpha-hemoglobin with modifications targeted exclusively to amino groups: functional and structural advantages of free Cys-93(beta) in the PEG-Hb adduct.

PubMed

Li, Dongxia; Hu, Tao; Manjula, Belur N; Acharya, Seetharama A

2009-11-01

Cys-93(beta) of hemoglobin (Hb) was reversibly protected as a mixed disulfide with thiopyridine during extension arm facilitated (EAF) PEGylation and its influence on the structural and functional properties of the EAF-PEG-Hb has been investigated. Avoiding PEGylation of Cys-93(beta) in the EAF-PEG-Hb lowers the level of perturbation of heme pocket, alpha1beta2 interface, autoxidation, heme loss, and the O(2) affinity, as compared to the EAF-PEG-Hb with PEGylation of Cys-93(beta).The structural and functional advantages of reversible protection of Cys-93(beta) during EAF PEGylation of oxy-Hb has been compared with Euro PEG-Hb generated by EAF PEGylation of deoxy Hb where Cys-93(beta) is free in the final product. The alphaalpha-fumaryl cross-linking and EAF PEGylation targeted exclusively to Lys residues has been combined together for generation of second-generation EAF-PEG-Hb with lower oxygen affinity. The PEG chains engineered on Lys as well as PEGylation of Cys-93(beta) independently contribute to the stabilization of oxy conformation of Hb and hence increase the oxygen affinity of Hb. However, oxygen affinity of the EAF-PEG-alphaalpha-Hb is more sensitive to the presence of PEGylation on Cys-93(beta) than that of the EAF-PEG-Hb. The present modified EAF PEGylation platform is expected to facilitate the design of novel versions of the EAF-PEG-Hbs that can now integrate the advantages of avoiding PEGylation of Cys-93(beta).

A new 68Ge/68Ga generator system using an organic polymer containing N-methylglucamine groups as adsorbent for 68Ge.

PubMed

Nakayama, M; Haratake, M; Ono, M; Koiso, T; Harada, K; Nakayama, H; Yahara, S; Ohmomo, Y; Arano, Y

2003-01-01

A macroporous styrene-divinylbenzene copolymer containing N-methylglucamine groups was selected for a new 68Ge/68Ga generator system. This resin packed into a column effectively adsorbed the parent nuclide 68Ge. The daughter 68Ga was eluted from the resin with a solution of a low-affinity gallium chelating ligand such as citric or phosphoric acid. The 68Ge leakage was less than 0.0004% of the 68Ge adsorbed on the resin. By simple mixing of transferrin and desferoxamine conjugated HSA and IgG with the eluate from the column, 68Ga-labeling was completed in high yield. Copyright 2002 Elsevier Science Ltd.

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

PubMed

Seguin, L; Widdowson, P S; Giesen-Crouse, E

1992-12-01

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

[Progresses in screening active compounds from herbal medicine by affinity chromatography].

PubMed

Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

2015-03-01

Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening.

[Immunogenicity and safety of the influenza vaccine, in a population older than 55-years in Mexico].

PubMed

Ayala-Montiel, Octavio; Mascareñas, César; García-Hernández, Delfino; Rendón-Muñiz, Jorge; López, Irma; Felipe Montaño, Luis; Zenteno, I; Franco-Paredes, C

2005-01-01

To confirm the immunogenicity and tolerance of the inactivated, fractionated, and purified influenza vaccine, in a Mexican adult population aged 55 and older, medically served at a Petróleos Mexicanos Hospital (Pemex, Mexican Oil Company). The study was conducted between November and December, 2000, among ninety adult subjects aged 55 years and older who were seen at the Hospital Central Sur Pemex. The primary endpoints regarding immunogenicity were the percentage of individuals with protective antibodies targeting hemagglutinins higher than or equal to 1:40, and the percentage of subjects who seroconverted as measured by a four-fold increase in protective antibody production. Secondary endpoints included the frequency of local and systemic reactions to the vaccine. An additional criterion that was evaluated included antigen-antibody affinity assays to measure the polyclonal antibody response to the vaccine and the specific generation of high-affinity antibodies to viral proteins, before and after vaccination. The antibody protection rate was 95.6% against the HINI strain, 98.9% against the H3N2 strain, and a 100% against the B/Yamanashi strain. Seroconversion to the HINI strain was elicited in 74.4% of subjects, to the H3N2 strain in 88.9%, and to the B/Yamanashi strain in 82.2%. Eighteen (20%) subjects developed local reactions; 17 (18.8%) developed a systemic reaction post vaccination at day 5 and nine subjects (10%) at day 28. Local reactions consisted of pain in 10 (11.1%) subjects, redness in 8 (8.8%), and induration in 6 (6.6%). General malaise, headache, and fever were identified in 10, 8.8, and 0% of subjects, respectively, at day 5, and in 4.4, 6.6, and 0%, respectively, at day 28. Influenza vaccine was highly immunogenic in a healthy Mexican adult population aged 55 years and older. The generation of high-affinity antibodies to the virus after vaccination was also demonstrated. Local and systemic adverse reactions to the vaccine identified in our study were similar to those in previous reports. The results of this study can be extrapolated to other health institutions serving this adult population to increase influenza vaccine coverage rates.

Cloning, heterologous expression, and in situ characterization of the first high affinity nucleobase transporter from a protozoan.

PubMed

Burchmore, Richard J S; Wallace, Lynsey J M; Candlish, Denise; Al-Salabi, Mohammed I; Beal, Paul R; Barrett, Michael P; Baldwin, Stephen A; de Koning, Harry P

2003-06-27

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1.

PubMed

Real Hernandez, Luis M; Gonzalez de Mejia, Elvira

2017-04-01

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption. Copyright © 2017 Elsevier Inc. All rights reserved.

Alternate working fluids for solar air conditioning applications

NASA Technical Reports Server (NTRS)

Evans, R. D.; Beck, J. K.

1978-01-01

An experimental investigation of sixteen different refrigerant-absorbent fluid pairs has been carried out in order to determine their suitability as the working fluid in a solar-powered absorption cycle air conditioner. The criteria used in the initial selection of a refrigerant-absorbent pair included: high affinity (large negative deviation from Raoult's Law), high solubility, low specific heat, low viscosity, stability, corrosive properties, safety, and cost. For practical solar considerations of a fluid pair, refrigerants were selected with low boiling points whereas absorbent fluids were selected with a boiling point considerably above that of the refrigerant. Additional restrictions are determined by the operating temperatures of the absorber and the generator; these temperatures were specified as 100 F (39 C) and 170 F (77 C). Data are presented for a few selected pressures at the specified absorber and generator temperatures.

Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide-Protease Fusions.

PubMed

Sørensen, Hans Peter; Xu, Peng; Jiang, Longguang; Kromann-Hansen, Tobias; Jensen, Knud J; Huang, Mingdong; Andreasen, Peter A

2015-09-25

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human llamas: adaptation to altitude in subjects with high hemoglobin oxygen affinity.

PubMed Central

Hebbel, R P; Eaton, J W; Kronenberg, R S; Zanjani, E D; Moore, L G; Berger, E M

1978-01-01

To assess the adaptive value of the right-shift of the oxyhemoglobin dissociation curve (decreased affinity for oxygen) observed in humans upon altitude exposure, the short-term physiologic responses to altitude-induced hypoxia were evaluated in two subjects with a high oxygen affinity hemoglobin (Hb Andrew-Minneapolis) and in two of their normal siblings. In striking contrast to normal subjects, at moderately high altitude (3,100 m) the high affinity subjects manifested: (a) lesser increments in resting heart rate; (b) minimal increases in plasma and urinary erythropoietin; (c) no decrement in maximal oxygen consumption; and (d) no thrombocytopenia. There was no difference between subject pairs in 2,3-diphosphoglycerate response to altitude exposure. These results tend to contradict the belief that a decrease in hemoglobin oxygen affinity is of adaptive value to humans at moderate altitudes. Rather, they support the hypothesis that, despite disadvantages at low altitude, a left-shifted oxyhemoglobin dissociation curve may confer a degree of preadaptation to altitude. PMID:29054

Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria ▿†

PubMed Central

Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

2011-01-01

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)−1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur. PMID:21742924

Evolution of substrate specificity for the bile salt transporter ASBT (SLC10A2)[S

PubMed Central

Lionarons, Daniël A.; Boyer, James L.; Cai, Shi-Ying

2012-01-01

The apical Na+-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution. PMID:22669917

The Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus†

PubMed Central

Quinlan, R. Jason; Reinhart, Gregory D.

2008-01-01

Differences between the crystal structures of inhibitor-bound and uninihibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits containing two different extrinsic fluorophores simultaneously in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel Fluorescence Correlation Spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor-binding, as binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated. PMID:16981693

Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2

PubMed Central

Epa, V. Chandana; Dolezal, Olan; Doughty, Larissa; Xiao, Xiaowen; Jost, Christian; Plückthun, Andreas; Adams, Timothy E.

2013-01-01

Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84–1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions. PMID:23527120

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse—Methamphetamine and 3,4-methylenedioxymethamphetamine

PubMed Central

Celikel, Reha; Peterson, Eric C; Owens, S Michael; Varughese, Kottayil I

2009-01-01

Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(+)-METH therapeutic single chain antibody fragment (scFv6H4, KD= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name “ecstasy.” The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a C—H⋯O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 Å, and 2.4 Å, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization. PMID:19760665

Analysis of conjugation of chloramphenicol and hemoglobin by fluorescence, circular dichroism and molecular modeling

NASA Astrophysics Data System (ADS)

Ding, Fei; Liu, Wei; Sun, Ye; Yang, Xin-Ling; Sun, Ying; Zhang, Li

2012-01-01

Chloramphenicol is a low cost, broad spectrum, highly active antibiotic, and widely used in the treatment of serious infections, including typhoid fever and other life-threatening infections of the central nervous system and respiratory tract. The purpose of the present study was to examine the conjugation of chloramphenicol with hemoglobin (Hb) and compared with albumin at molecular level, utilizing fluorescence, UV/vis absorption, circular dichroism (CD) as well as molecular modeling. Fluorescence data indicate that drug bind Hb generate quenching via static mechanism, this corroborates UV/vis absorption measurements that the ground state complex formation with an affinity of 10 4 M -1, and the driving forces in the Hb-drug complex are hydrophilic interactions and hydrogen bonds, as derived from computational model. The accurate binding site of drug has been identified from the analysis of fluorescence and molecular modeling, α1β2 interface of Hb was assigned to possess high-affinity for drug, which located at the β-37 Trp nearby. The structural investigation of the complexed Hb by synchronous fluorescence, UV/vis absorption, and CD observations revealed some degree of Hb structure unfolding upon complexation. Based on molecular modeling, we can draw the conclusion that the binding affinity of drug with albumin is superior, compared with Hb. These phenomena can provide salient information on the absorption, distribution, pharmacology, and toxicity of chloramphenicol and other drugs which have analogous configuration with chloramphenicol.

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-Methamphetamine and 3,4-methylenedioxymethamphetamine.

PubMed

Celikel, Reha; Peterson, Eric C; Owens, S Michael; Varughese, Kottayil I

2009-11-01

Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(+)-METH therapeutic single chain antibody fragment (scFv6H4, K(D)= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name "ecstasy." The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a C--H...O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 A, and 2.4 A, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.

A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification.

PubMed

Fernandes, Cláudia S M; Pina, Ana Sofia; Dias, Ana M G C; Branco, Ricardo J F; Roque, Ana Cecília Afonso

2014-09-30

The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Computational redesign of a protein-protein interface for high affinity and binding specificity using modular architecture and naturally occurring template fragments.

PubMed

Potapov, V; Reichmann, D; Abramovich, R; Filchtinski, D; Zohar, N; Ben Halevy, D; Edelman, M; Sobolev, V; Schreiber, G

2008-12-05

A new method is presented for the redesign of protein-protein interfaces, resulting in specificity of the designed pair while maintaining high affinity. The design is based on modular interface architecture and was carried out on the interaction between TEM1 beta-lactamase and its inhibitor protein, beta-lactamase inhibitor protein. The interface between these two proteins is composed of several mostly independent modules. We previously showed that it is possible to delete a complete module without affecting the overall structure of the interface. Here, we replace a complete module with structure fragments taken from nonrelated proteins. Nature-optimized fragments were chosen from 10(7) starting templates found in the Protein Data Bank. A procedure was then developed to identify sets of interacting template residues with a backbone arrangement mimicking the original module. This generated a final list of 361 putative replacement modules that were ranked using a novel scoring function based on grouped atom-atom contact surface areas. The top-ranked designed complex exhibited an affinity of at least the wild-type level and a mode of binding that was remarkably specific despite the absence of negative design in the procedure. In retrospect, the combined application of three factors led to the success of the design approach: utilizing the modular construction of the interface, capitalizing on native rather than artificial templates, and ranking with an accurate atom-atom contact surface scoring function.

Hemoglobin–oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend?

PubMed Central

2016-01-01

ABSTRACT In air-breathing vertebrates at high altitude, fine-tuned adjustments in hemoglobin (Hb)–O2 affinity provide an energetically efficient means of mitigating the effects of arterial hypoxemia. However, it is not always clear whether an increased or decreased Hb–O2 affinity should be expected to improve tissue O2 delivery under different degrees of hypoxia, due to the inherent trade-off between arterial O2 loading and peripheral O2 unloading. Theoretical results indicate that the optimal Hb–O2 affinity varies as a non-linear function of environmental O2 availability, and the threshold elevation at which an increased Hb–O2 affinity becomes advantageous depends on the magnitude of diffusion limitation (the extent to which O2 equilibration at the blood–gas interface is limited by the kinetics of O2 exchange). This body of theory provides a framework for interpreting the possible adaptive significance of evolved changes in Hb–O2 affinity in vertebrates that have colonized high-altitude environments. To evaluate the evidence for an empirical generalization and to test theoretical predictions, I synthesized comparative data in a phylogenetic framework to assess the strength of the relationship between Hb–O2 affinity and native elevation in mammals and birds. Evidence for a general trend in mammals is equivocal, but there is a remarkably strong positive relationship between Hb–O2 affinity and native elevation in birds. Evolved changes in Hb function in high-altitude birds provide one of the most compelling examples of convergent biochemical adaptation in vertebrates. PMID:27802149

Hemoglobin-oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend?

PubMed

Storz, Jay F

2016-10-15

In air-breathing vertebrates at high altitude, fine-tuned adjustments in hemoglobin (Hb)-O 2 affinity provide an energetically efficient means of mitigating the effects of arterial hypoxemia. However, it is not always clear whether an increased or decreased Hb-O 2 affinity should be expected to improve tissue O 2 delivery under different degrees of hypoxia, due to the inherent trade-off between arterial O 2 loading and peripheral O 2 unloading. Theoretical results indicate that the optimal Hb-O 2 affinity varies as a non-linear function of environmental O 2 availability, and the threshold elevation at which an increased Hb-O 2 affinity becomes advantageous depends on the magnitude of diffusion limitation (the extent to which O 2 equilibration at the blood-gas interface is limited by the kinetics of O 2 exchange). This body of theory provides a framework for interpreting the possible adaptive significance of evolved changes in Hb-O 2 affinity in vertebrates that have colonized high-altitude environments. To evaluate the evidence for an empirical generalization and to test theoretical predictions, I synthesized comparative data in a phylogenetic framework to assess the strength of the relationship between Hb-O 2 affinity and native elevation in mammals and birds. Evidence for a general trend in mammals is equivocal, but there is a remarkably strong positive relationship between Hb-O 2 affinity and native elevation in birds. Evolved changes in Hb function in high-altitude birds provide one of the most compelling examples of convergent biochemical adaptation in vertebrates. © 2016. Published by The Company of Biologists Ltd.

Specificity of cell–cell adhesion by classical cadherins: Critical role for low-affinity dimerization through β-strand swapping

PubMed Central

Chen, Chien Peter; Posy, Shoshana; Ben-Shaul, Avinoam; Shapiro, Lawrence; Honig, Barry H.

2005-01-01

Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cell–cell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by “swapping” the N-terminal β-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that “closed” monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell–cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems. PMID:15937105

DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

PubMed

Bitaraf, F S; Rasooli, I; Mousavi Gargari, S L

2016-03-01

Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

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

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

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

1988-05-01

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

Simulation of NO and O2 transport facilitated by polymerized hemoglobin solutions in an arteriole that takes into account wall shear stress-induced NO production.

PubMed

Zhou, Yipin; Cabrales, Pedro; Palmer, Andre F

2012-03-01

A mathematical model was developed to study nitric oxide (NO) and oxygen (O(2)) transport in an arteriole and surrounding tissues exposed to a mixture of red blood cells (RBCs) and hemoglobin (Hb)-based O(2) carriers (HBOCs). A unique feature of this model is the inclusion of blood vessel wall shear stress-induced production of endothelial-derived NO, which is very sensitive to the viscosity of the RBC and HBOC mixture traversing the blood vessel lumen. Therefore in this study, a series of polymerized bovine Hb (PolyHb) solutions with high viscosity, varying O(2) affinities, NO dioxygenation rate constants and O(2) dissociation rate constants that were previously synthesized and characterized by our group was evaluated via mathematical modeling, in order to investigate the effect of these biophysical properties on the transport of NO and O(2) in an arteriole and its surrounding tissues subjected to anemia with the commercial HBOC Oxyglobin® and cell-free bovine Hb (bHb) serving as appropriate controls. The computer simulation results indicated that transfusion of high viscosity PolyHb solutions promoted blood vessel wall shear stress dependent generation of the vasodilator NO, especially in the blood vessel wall and should transport enough NO inside the smooth muscle layer to activate vasodilation compared to the commercial HBOC Oxyglobin® and cell-free bHb. However, NO scavenging in the arteriole lumen was unavoidable due to the intrinsic high NO dioxygenation rate constant of the HBOCs being studied. This study also observed that all PolyHbs could potentially improve tissue oxygenation under hypoxic conditions, while low O(2) affinity PolyHbs were more effective in oxygenating tissues under normoxic conditions compared with high O(2) affinity PolyHbs. In addition, all ultrahigh molecular weight PolyHbs displayed higher O(2) transfer rates than the commercial HBOC Oxyglobin® and cell-free bHb. Therefore, these results suggest that ultrahigh molecular weight PolyHb solutions could be used as safe and efficacious O(2) carriers for use in transfusion medicine. It also suggests that future generations of PolyHb solutions should possess lower NO dioxygenation reaction rate constants in order to reduce NO scavenging, while maintaining high solution viscosity to take advantage of wall shear stress-induced NO production. Taken together, we suggest that this mathematical model can be used to predict the vasoactivity of HBOCs and help guide the design and optimization of the next generation of HBOCs for use in transfusion medicine. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterization of nicotine binding to the rat brain P/sub 2/ preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

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

Sloan, J.W.

1984-01-01

These studies show that nicotine binds to the rat brain P/sub 2/ preparation by saturable and reversible processes. Multiple binding sites were revealed by the configuration of saturation, kinetic and Scatchard plots. A least squares best fit of Scatchard data using nonlinear curve fitting programs confirmed the presence of a very high affinity site, an up-regulatory site, a high affinity site and one or two low affinity sites. Stereospecificity was demonstrated for the up-regulatory site where (+)-nicotine was more effective and for the high affinity site where (-)-nicotine had a higher affinity. Drugs which selectively up-regulate nicotine binding site(s) havemore » been identified. Further, separate very high and high affinity sites were identified for (-)- and (+)-(/sup 3/H)nicotine, based on evidence that the site density for the (-)-isomer is 10 times greater than that for the (+)-isomer at these sites. Enhanced nicotine binding has been shown to be a statistically significant phenomenon which appears to be a consequence of drugs binding to specific site(s) which up-regulate binding at other site(s). Although Scatchard and Hill plots indicate positive cooperatively, up-regulation more adequately describes the function of these site(s). A separate up-regulatory site is suggested by the following: (1) Drugs vary markedly in their ability to up-regulate binding. (2) Both the affinity and the degree of up-regulation can be altered by structural changes in ligands. (3) Drugs with specificity for up-regulation have been identified. (4) Some drugs enhance binding in a dose-related manner. (5) Competition studies employing cold (-)- and (+)-nicotine against (-)- and (+)-(/sup 3/H)nicotine show that the isomers bind to separate sites which up-regulate binding at the (-)- and (+)-nicotine high affinity sites and in this regard (+)-nicotine is more specific and efficacious than (-)-nicotine.« less

Independent-particle models for light negative atomic ions

NASA Technical Reports Server (NTRS)

Ganas, P. S.; Talman, J. D.; Green, A. E. S.

1980-01-01

For the purposes of astrophysical, aeronomical, and laboratory application, a precise independent-particle model for electrons in negative atomic ions of the second and third period is discussed. The optimum-potential model (OPM) of Talman et al. (1979) is first used to generate numerical potentials for eight of these ions. Results for total energies and electron affinities are found to be very close to Hartree-Fock solutions. However, the OPM and HF electron affinities both depart significantly from experimental affinities. For this reason, two analytic potentials are developed whose inner energy levels are very close to the OPM and HF levels but whose last electron eigenvalues are adjusted precisely with the magnitudes of experimental affinities. These models are: (1) a four-parameter analytic characterization of the OPM potential and (2) a two-parameter potential model of the Green, Sellin, Zachor type. The system O(-) or e-O, which is important in upper atmospheric physics is examined in some detail.

A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding.

PubMed

Mercado, R; Hernández, J

1992-09-18

Axonal growth cone particles (AGCP) isolated from prenatal and postnatal rat brain had different high-affinity 5-HT uptake characteristics. In postnatal AGCP the uptake behaves as in the adult rat brain, while in the prenatal AGCP the uptake characteristics seem to be in a transitional stage. Also in prenatal AGCP we observed specific, high-affinity 5-HT binding sites. These results support the idea of an important role for 5-HT during axogenesis.

Novel, customizable scoring functions, parameterized using N-PLS, for structure-based drug discovery.

PubMed

Catana, Cornel; Stouten, Pieter F W

2007-01-01

The ability to accurately predict biological affinity on the basis of in silico docking to a protein target remains a challenging goal in the CADD arena. Typically, "standard" scoring functions have been employed that use the calculated docking result and a set of empirical parameters to calculate a predicted binding affinity. To improve on this, we are exploring novel strategies for rapidly developing and tuning "customized" scoring functions tailored to a specific need. In the present work, three such customized scoring functions were developed using a set of 129 high-resolution protein-ligand crystal structures with measured Ki values. The functions were parametrized using N-PLS (N-way partial least squares), a multivariate technique well-known in the 3D quantitative structure-activity relationship field. A modest correlation between observed and calculated pKi values using a standard scoring function (r2 = 0.5) could be improved to 0.8 when a customized scoring function was applied. To mimic a more realistic scenario, a second scoring function was developed, not based on crystal structures but exclusively on several binding poses generated with the Flo+ docking program. Finally, a validation study was conducted by generating a third scoring function with 99 randomly selected complexes from the 129 as a training set and predicting pKi values for a test set that comprised the remaining 30 complexes. Training and test set r2 values were 0.77 and 0.78, respectively. These results indicate that, even without direct structural information, predictive customized scoring functions can be developed using N-PLS, and this approach holds significant potential as a general procedure for predicting binding affinity on the basis of in silico docking.

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

PubMed

Sueck, Franziska; Poór, Miklós; Faisal, Zelma; Gertzen, Christoph G W; Cramer, Benedikt; Lemli, Beáta; Kunsági-Máté, Sándor; Gohlke, Holger; Humpf, Hans-Ulrich

2018-06-22

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus . 2′ R -Ochratoxin A (2′ R -OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′ R -OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′ R -OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′ R -OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′ R -OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a log K of 7.0⁻7.6 was calculated, while for its thermally produced isomer 2′ R -OTA, a lower, but still high, log K of 6.2⁻6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′ R -OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′ R -OTA found in human blood samples.

Epidermal growth factor receptor-targeted lipid nanoparticles retain self-assembled nanostructures and provide high specificity

NASA Astrophysics Data System (ADS)

Zhai, Jiali; Scoble, Judith A.; Li, Nan; Lovrecz, George; Waddington, Lynne J.; Tran, Nhiem; Muir, Benjamin W.; Coia, Gregory; Kirby, Nigel; Drummond, Calum J.; Mulet, Xavier

2015-02-01

Next generation drug delivery utilising nanoparticles incorporates active targeting to specific sites. In this work, we combined targeting with the inherent advantages of self-assembled lipid nanoparticles containing internal nano-structures. Epidermal growth factor receptor (EGFR)-targeting, PEGylated lipid nanoparticles using phytantriol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-maleimide amphiphiles were created. The self-assembled lipid nanoparticles presented here have internal lyotropic liquid crystalline nano-structures, verified by synchrotron small angle X-ray scattering and cryo-transmission electron microscopy, that offer the potential of high drug loading and enhanced cell penetration. Anti-EGFR Fab' fragments were conjugated to the surface of nanoparticles via a maleimide-thiol reaction at a high conjugation efficiency and retained specificity following conjugation to the nanoparticles. The conjugated nanoparticles were demonstrated to have high affinity for an EGFR target in a ligand binding assay.Next generation drug delivery utilising nanoparticles incorporates active targeting to specific sites. In this work, we combined targeting with the inherent advantages of self-assembled lipid nanoparticles containing internal nano-structures. Epidermal growth factor receptor (EGFR)-targeting, PEGylated lipid nanoparticles using phytantriol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-maleimide amphiphiles were created. The self-assembled lipid nanoparticles presented here have internal lyotropic liquid crystalline nano-structures, verified by synchrotron small angle X-ray scattering and cryo-transmission electron microscopy, that offer the potential of high drug loading and enhanced cell penetration. Anti-EGFR Fab' fragments were conjugated to the surface of nanoparticles via a maleimide-thiol reaction at a high conjugation efficiency and retained specificity following conjugation to the nanoparticles. The conjugated nanoparticles were demonstrated to have high affinity for an EGFR target in a ligand binding assay. Electronic supplementary information (ESI) available: Fig. S1-S4. See DOI: 10.1039/c4nr05200e

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

PubMed

Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

2004-03-07

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

Inhibition of the GAS6/AXL pathway augments the efficacy of chemotherapies

DOE PAGES

Kariolis, Mihalis S.; Miao, Yu Rebecca; Diep, Anh; ...

2016-11-28

The AXL receptor and its activating ligand, growth arrest–specific 6 (GAS6), are important drivers of metastasis and therapeutic resistance in human cancers. Given the critical roles that GAS6 and AXL play in refractory disease, this signaling axis represents an attractive target for therapeutic intervention. But, the strong picomolar binding affinity between GAS6 and AXL and the promiscuity of small molecule inhibitors represent important challenges faced by current anti-AXL therapeutics. We have addressed these obstacles by engineering a second-generation, high-affinity AXL decoy receptor with an apparent affinity of 93 femtomolar to GAS6. Our decoy receptor, MYD1-72, profoundly inhibited disease progression inmore » aggressive preclinical models of human cancers and induced cell killing in leukemia cells. When directly compared with the most advanced anti-AXL small molecules in the clinic, MYD1-72 achieved superior antitumor efficacy while displaying no toxicity. Furthermore, we uncovered a relationship between AXL and the cellular response to DNA damage whereby abrogation of AXL signaling leads to accumulation of the DNA-damage markers γH2AX, 53BP1, and RAD51. MYD1-72 exploited this relationship, leading to improvements upon the therapeutic index of current standard-of-care chemotherapies in preclinical models of advanced pancreatic and ovarian cancer.« less

Design-Based Peptidomimetic Ligand Discovery to Target HIV TAR RNA Using Comparative Analysis of Different Docking Methods.

PubMed

Fu, Junjie; Xia, Amy; Dai, Yao; Qi, Xin

2016-01-01

Discovering molecules capable of binding to HIV trans-activation responsive region (TAR) RNA thereby disrupting its interaction with Tat protein is an attractive strategy for developing novel antiviral drugs. Computational docking is considered as a useful tool for predicting binding affinity and conducting virtual screening. Although great progress in predicting protein-ligand interactions has been achieved in the past few decades, modeling RNA-ligand interactions is still largely unexplored due to the highly flexible nature of RNA. In this work, we performed molecular docking study with HIV TAR RNA using previously identified cyclic peptide L22 and its analogues with varying affinities toward HIV-1 TAR RNA. Furthermore, sarcosine scan was conducted to generate derivatives of CGP64222, a peptide-peptoid hybrid with inhibitory activity on Tat/TAR RNA interaction. Each compound was docked using CDOCKER, Surflex-Dock and FlexiDock to compare the effectiveness of each method. It was found that FlexiDock energy values correlated well with the experimental Kd values and could be used to predict the affinity of the ligands toward HIV-1 TAR RNA with a superior accuracy. Our results based on comparative analysis of different docking methods in RNA-ligand modeling will facilitate the structure-based discovery of HIV TAR RNA ligands for antiviral therapy.

Inhibition of the GAS6/AXL pathway augments the efficacy of chemotherapies

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

Kariolis, Mihalis S.; Miao, Yu Rebecca; Diep, Anh

The AXL receptor and its activating ligand, growth arrest–specific 6 (GAS6), are important drivers of metastasis and therapeutic resistance in human cancers. Given the critical roles that GAS6 and AXL play in refractory disease, this signaling axis represents an attractive target for therapeutic intervention. But, the strong picomolar binding affinity between GAS6 and AXL and the promiscuity of small molecule inhibitors represent important challenges faced by current anti-AXL therapeutics. We have addressed these obstacles by engineering a second-generation, high-affinity AXL decoy receptor with an apparent affinity of 93 femtomolar to GAS6. Our decoy receptor, MYD1-72, profoundly inhibited disease progression inmore » aggressive preclinical models of human cancers and induced cell killing in leukemia cells. When directly compared with the most advanced anti-AXL small molecules in the clinic, MYD1-72 achieved superior antitumor efficacy while displaying no toxicity. Furthermore, we uncovered a relationship between AXL and the cellular response to DNA damage whereby abrogation of AXL signaling leads to accumulation of the DNA-damage markers γH2AX, 53BP1, and RAD51. MYD1-72 exploited this relationship, leading to improvements upon the therapeutic index of current standard-of-care chemotherapies in preclinical models of advanced pancreatic and ovarian cancer.« less

Haemoglobin Pierre-Benite--a high affinity variant associated with relative polycythaemia.

PubMed

Beard, M E; Potter, H C; Spearing, R L; Brennan, S O

2001-12-01

This is the second reported example of Hb Pierre--Benite (beta90 Glu-->Asp). This mutation is associated with increased oxygen affinity and polycythaemia. No instability was found and there was no charge shift detected by cellulose acetate electrophoresis at pH 8.3. The mutation was however, clearly indicated by electrospray ionization mass spectrometry (ESI MS), which showed an abnormal beta chain with a 14 Da decrease in mass. Blood volume studies documented a relative rather than a true polycythaemia and this finding has been reported in at least two other high affinity haemoglobin variants--Hb Heathrow and Hb Rahere. This finding led to delay in diagnosis because high oxygen affinity variants are conventionally considered to cause a true polycythaemia.

Tensor scale-based fuzzy connectedness image segmentation

NASA Astrophysics Data System (ADS)

Saha, Punam K.; Udupa, Jayaram K.

2003-05-01

Tangible solutions to image segmentation are vital in many medical imaging applications. Toward this goal, a framework based on fuzzy connectedness was developed in our laboratory. A fundamental notion called "affinity" - a local fuzzy hanging togetherness relation on voxels - determines the effectiveness of this segmentation framework in real applications. In this paper, we introduce the notion of "tensor scale" - a recently developed local morphometric parameter - in affinity definition and study its effectiveness. Although, our previous notion of "local scale" using the spherical model successfully incorporated local structure size into affinity and resulted in measureable improvements in segmentation results, a major limitation of the previous approach was that it ignored local structural orientation and anisotropy. The current approach of using tensor scale in affinity computation allows an effective utilization of local size, orientation, and ansiotropy in a unified manner. Tensor scale is used for computing both the homogeneity- and object-feature-based components of affinity. Preliminary results of the proposed method on several medical images and computer generated phantoms of realistic shapes are presented. Further extensions of this work are discussed.

Specificity and Affinity Quantification of Flexible Recognition from Underlying Energy Landscape Topography

PubMed Central

Chu, Xiakun; Wang, Jin

2014-01-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition. PMID:25144525

Specificity and affinity quantification of flexible recognition from underlying energy landscape topography.

PubMed

Chu, Xiakun; Wang, Jin

2014-08-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition.

Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

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

Senogles, S.E.; Caron, M.G.

1986-05-01

The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..Smore » binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.« less

Molecular Basis of Autophagic Cell Death in Prostate Cancer

DTIC Science & Technology

2009-03-01

lipid-binding proteinwith high affinity for phosphatidic acid (PA) and cardiolipin (CL). Previously, it has been shown that PA directly interacted...lyceride), PI (phosphatidylinositol), DAG (diacylglycerol), PI4P (PtdIns(4)P), PA ( phosphatidic acid ), PI4,5P2 (PtdIns(4,5)P2), PS (phosphatidylserine...with high affinity for phosphatidic acid and cardiolipin and less affinity for various phosphoinositides. Functional genomics analysis identified 5

Efficient T-cell receptor signaling requires a high-affinity interaction between the Gads C-SH3 domain and the SLP-76 RxxK motif.

PubMed

Seet, Bruce T; Berry, Donna M; Maltzman, Jonathan S; Shabason, Jacob; Raina, Monica; Koretzky, Gary A; McGlade, C Jane; Pawson, Tony

2007-02-07

The relationship between the binding affinity and specificity of modular interaction domains is potentially important in determining biological signaling responses. In signaling from the T-cell receptor (TCR), the Gads C-terminal SH3 domain binds a core RxxK sequence motif in the SLP-76 scaffold. We show that residues surrounding this motif are largely optimized for binding the Gads C-SH3 domain resulting in a high-affinity interaction (K(D)=8-20 nM) that is essential for efficient TCR signaling in Jurkat T cells, since Gads-mediated signaling declines with decreasing affinity. Furthermore, the SLP-76 RxxK motif has evolved a very high specificity for the Gads C-SH3 domain. However, TCR signaling in Jurkat cells is tolerant of potential SLP-76 crossreactivity, provided that very high-affinity binding to the Gads C-SH3 domain is maintained. These data provide a quantitative argument that the affinity of the Gads C-SH3 domain for SLP-76 is physiologically important and suggest that the integrity of TCR signaling in vivo is sustained both by strong selection of SLP-76 for the Gads C-SH3 domain and by a capacity to buffer intrinsic crossreactivity.

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

PubMed

Heng, Sabrina; Stieglitz, Kimberly A; Eldo, Joby; Xia, Jiarong; Cardia, James P; Kantrowitz, Evan R

2006-08-22

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

PubMed

Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C; Munson, P J; Rodbard, D

1979-12-01

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.

Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

PubMed Central

Haylock, Anna-Karin; Nilvebrant, Johan; Mortensen, Anja; Velikyan, Irina; Nestor, Marika; Falk, Ronny

2017-01-01

Aim The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients. Materials and methods Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I. Results Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor. Conclusion The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging. PMID:29029420

Hb Potomac (101 Glu replaced by Asp): speculations on placental oxygen transport in carriers of high-affinity hemoglobins.

PubMed

Charache, S; Jacobson, R; Brimhall, B; Murphy, E A; Hathaway, P; Winslow, R; Jones, R; Rath, C; Simkovich, J

1978-02-01

Blood from a woman with unexplained erythrocytosis had increased oxygen affinity, but no abnormality could be detected by electrophoresis or chromatography of her hemolysate. Separation of the tryptic peptides of her beta chains disclosed two half-sized peaks in the regions of beta T-11. The faster of these was abnormal, with the structure beta 101 Glu replaced by Asp. The new hemoglobin was called "Potomac." Three of the proband's four surviving siblings and both of her children were carriers. Differences in the ratio of carrier: normal children born to male of female carriers of 23 other high-affinity hemoglobins were not significant. The high proportion of carriers in this kindred was probably due to chance alone, and not because high maternal oxygen affinity interfered with oxygen transport to fetuses with normal hemoglobin.

Biomarker Candidates of Chlamydophila pneumoniae Proteins and Protein Fragments Identified by Affinity-Proteomics Using FTICR-MS and LC-MS/MS

NASA Astrophysics Data System (ADS)

Susnea, Iuliana; Bunk, Sebastian; Wendel, Albrecht; Hermann, Corinna; Przybylski, Michael

2011-04-01

We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.

Lesion-induced plasticity of high affinity choline uptake in the developing rat fascia dentata.

PubMed

Nadler, J V; Shelton, D L; Cotman, C W

1979-03-23

After removal of the perforant path input to the rat fascia dentata at the age of 11 days, cholinergic septohippocampal fibers invade the denervated area. We have examined the effect of this lesion on hemicholinium-sensitive, high affinity choline uptake and its coupling to acetylcholine synthesis, specific properties of the septohippocampal input. Removal of the ipsilateral perforant path fibers increased the velocity of high affinity choline uptake by dentate particulate preparations, usually within 1 day. Studies conducted 5--104 days after operation showed a consistent 50--65% elevation in the molecular (denervated) layer. In contrast, the choline uptake rate in the granular layer eventually decreased slightly. Calculation of choline uptake rates independently of protein (per whole region) revealed that fasciae dentatae from operated and control sides accumulated choline at approximately equal rates, but on the operated side a greater percentage was transported by structures from the molecular layer and a lesser percentage by those from the granular layer. The rate of acetylcholine synthesis from exogenous choline increased to the same extent as high affinity choline uptake from 3 days after operation onwards. The changes in high affinity choline uptake and acetylcholine synthesis coincided spatially and temporally with the reactive growth of septohippocampal fibers. Our results support the view that a perforant path lesion during development permanently alters the distribution of functional septohippocampal boutons in the fascia dentata. Acetylcholine synthesis is regulated to the same extent by high affinity choline uptake in the anomalous boutons as in normally located boutons.

A putative role for the plasma membrane potential in the control of the expression of the gene encoding the tomato high-affinity potassium transporter HAK5.

PubMed

Nieves-Cordones, Manuel; Miller, Anthony J; Alemán, Fernando; Martínez, Vicente; Rubio, Francisco

2008-12-01

A chimeric CaHAK1-LeHAK5 transporter with only 15 amino acids of CaHAK1 in the N-terminus mediates high-affinity K(+) uptake in yeast cells. Kinetic and expression analyses strongly suggest that LeHAK5 mediates a significant proportion of the high-affinity K(+) uptake shown by K(+)-starved tomato (Solanum lycopersicum) plants. The development of high-affinity K(+) uptake, putatively mediated by LeHAK5, was correlated with increased LeHAK5 mRNA levels and a more negative electrical potential difference across the plasma membrane of root epidermal and cortical cells. However, this increase in high-affinity K(+) uptake was not correlated with the root K(+) content. Thus, (i) growth conditions that result in a hyperpolarized root plasma membrane potential, such as K(+) starvation or growth in the presence of NH(4) (+), but which do not decrease the K(+) content, lead to increased LeHAK5 expression; (ii) the presence of NaCl in the growth solution, which prevents the hyperpolarization induced by K(+) starvation, also prevents LeHAK5 expression. Moreover, once the gene is induced, depolarization of the plasma membrane potential then produces a decrease in the LeHAK5 mRNA. On the basis of these results, we propose that the plant membrane electrical potential plays a role in the regulation of the expression of this gene encoding a high-affinity K(+) transporter.

Targeted thermal therapy with genetically engineered magnetite magnetosomes@RGD: Photothermia is far more efficient than magnetic hyperthermia.

PubMed

Plan Sangnier, Anouchka; Preveral, Sandra; Curcio, Alberto; K A Silva, Amanda; Lefèvre, Chistopher T; Pignol, David; Lalatonne, Yoann; Wilhelm, Claire

2018-06-10

Providing appropriate means for heat generation by low intratumoral nanoparticle concentrations is a major challenge for cancer nanotherapy. Here we propose RGD-tagged magnetosomes (magnetosomes@RGD) as a biogenic, genetically engineered, inorganic platform for multivalent thermal cancer treatment. Magnetosomes@RGD are biomagnetite nanoparticles synthesized by genetically modified magnetotactic bacteria thanks to a translational fusion of the RGD peptide with the magnetosomal protein MamC. Magnetosomes@RGD thus combine the high crystallinity of their magnetite core with efficient surface functionalization. The specific affinity of RGD was first quantified by single-cell magnetophoresis with a variety of cell types, including immune, muscle, endothelial, stem and cancer cells. The highest affinity and cellular uptake was observed with PC3 prostatic and HeLa uterine cancer cells. The efficiency of photothermia and magnetic hyperthermia was then compared on PC3 cells. Unexpectedly, photothermia was far more efficient than magnetic hyperthermia, which was almost totally inhibited by the cellular environment. RGD targeting was then assessed in vivo at tumor site, in mice bearing PC3 tumors. As a result, we demonstrate that targeted magnetic nanoparticles could generate heat on a therapeutic level after systemic administration, but only under laser excitation, and successfully inhibit tumor progression. Copyright © 2018 Elsevier B.V. All rights reserved.

Multiplexed evaluation of capture agent binding kinetics using arrays of silicon photonic microring resonators.

PubMed

Byeon, Ji-Yeon; Bailey, Ryan C

2011-09-07

High affinity capture agents recognizing biomolecular targets are essential in the performance of many proteomic detection methods. Herein, we report the application of a label-free silicon photonic biomolecular analysis platform for simultaneously determining kinetic association and dissociation constants for two representative protein capture agents: a thrombin-binding DNA aptamer and an anti-thrombin monoclonal antibody. The scalability and inherent multiplexing capability of the technology make it an attractive platform for simultaneously evaluating the binding characteristics of multiple capture agents recognizing the same target antigen, and thus a tool complementary to emerging high-throughput capture agent generation strategies.

Binding of ACE-inhibitors to in vitro and patient-derived amyloid-β fibril models.

PubMed

Bhavaraju, Manikanthan; Phillips, Malachi; Bowman, Deborah; Aceves-Hernandez, Juan M; Hansmann, Ulrich H E

2016-01-07

Currently, no drugs exist that can prevent or reverse Alzheimer's disease, a neurodegenerative disease associated with the presence, in the brain, of plaques that are composed of β-amyloid (Aβ) peptides. Recent studies suggest that angiotensin-converting enzyme (ACE) inhibitors, a set of drugs used to treat hypertension, may inhibit amyloid formation in vitro. In the present study, we investigate through computer simulations the binding of ACE inhibitors to patient-derived Aβ fibrils and contrast it with that of ACE inhibitors binding to in vitro generated fibrils. The binding affinities of the ACE inhibitors are compared with that of Congo red, a dye that is used to identify amyloid structures and that is known to be a weak inhibitor of Aβ aggregation. We find that ACE inhibitors have a lower binding affinity to the patient-derived fibrils than to in vitro generated ones. For patient-derived fibrils, their binding affinities are even lower than that of Congo red. Our observations raise doubts on the hypothesis that these drugs inhibit fibril formation in Alzheimer patients by interacting directly with the amyloids.

Comparison of three methods for registration of abdominal/pelvic volume data sets from functional-anatomic scans

NASA Astrophysics Data System (ADS)

Mahmoud, Faaiza; Ton, Anthony; Crafoord, Joakim; Kramer, Elissa L.; Maguire, Gerald Q., Jr.; Noz, Marilyn E.; Zeleznik, Michael P.

2000-06-01

The purpose of this work was to evaluate three volumetric registration methods in terms of technique, user-friendliness and time requirements. CT and SPECT data from 11 patients were interactively registered using: a 3D method involving only affine transformation; a mixed 3D - 2D non-affine (warping) method; and a 3D non-affine (warping) method. In the first method representative isosurfaces are generated from the anatomical images. Registration proceeds through translation, rotation, and scaling in all three space variables. Resulting isosurfaces are fused and quantitative measurements are possible. In the second method, the 3D volumes are rendered co-planar by performing an oblique projection. Corresponding landmark pairs are chosen on matching axial slice sets. A polynomial warp is then applied. This method has undergone extensive validation and was used to evaluate the results. The third method employs visualization tools. The data model allows images to be localized within two separate volumes. Landmarks are chosen on separate slices. Polynomial warping coefficients are generated and data points from one volume are moved to the corresponding new positions. The two landmark methods were the least time consuming (10 to 30 minutes from start to finish), but did demand a good knowledge of anatomy. The affine method was tedious and required a fair understanding of 3D geometry.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

PubMed

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

2014-10-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

Predicting the Effect of Mutations on Protein-Protein Binding Interactions through Structure-Based Interface Profiles

PubMed Central

Brender, Jeffrey R.; Zhang, Yang

2015-01-01

The formation of protein-protein complexes is essential for proteins to perform their physiological functions in the cell. Mutations that prevent the proper formation of the correct complexes can have serious consequences for the associated cellular processes. Since experimental determination of protein-protein binding affinity remains difficult when performed on a large scale, computational methods for predicting the consequences of mutations on binding affinity are highly desirable. We show that a scoring function based on interface structure profiles collected from analogous protein-protein interactions in the PDB is a powerful predictor of protein binding affinity changes upon mutation. As a standalone feature, the differences between the interface profile score of the mutant and wild-type proteins has an accuracy equivalent to the best all-atom potentials, despite being two orders of magnitude faster once the profile has been constructed. Due to its unique sensitivity in collecting the evolutionary profiles of analogous binding interactions and the high speed of calculation, the interface profile score has additional advantages as a complementary feature to combine with physics-based potentials for improving the accuracy of composite scoring approaches. By incorporating the sequence-derived and residue-level coarse-grained potentials with the interface structure profile score, a composite model was constructed through the random forest training, which generates a Pearson correlation coefficient >0.8 between the predicted and observed binding free-energy changes upon mutation. This accuracy is comparable to, or outperforms in most cases, the current best methods, but does not require high-resolution full-atomic models of the mutant structures. The binding interface profiling approach should find useful application in human-disease mutation recognition and protein interface design studies. PMID:26506533

Evaluation of anti-sepsis activity by compounds with high affinity to lipid a from HuanglianJiedu decoction.

PubMed

Xu, Yubin; Guo, Song; Chen, Guirong; Zhang, Mingbo; Zhang, Xu; Dou, Deqiang

2017-12-01

HuanglianJiedu decoction (HJD) is a classic prescription for heat-clearing away and detoxifying, which is used for the clinical treatment of sepsis, due to sepsis refers to the systemic inflammatory response induced by infection in western medicine, and infection belongs to the category of poison-heat syndrome in traditional Chinese medicine. Previous study had elucidated the effective components from HJD with high affinity to lipid A, which can generate the release of pro-inflammatory-cytokines, resulting in sepsis. Now the anti-sepsis activities of these compounds were evaluated. Immunofluorescence, immunohistochemical staining, ELISA and MTT methods were used to evaluated these compounds. Immunofluorescence analysis evaluated the effects of compounds on the binding of FITC-LPS to RAW264.7 cells, and showed the fluorescence intensity was significant attenuated in geniposides, palmatine, baicalin and berberine groups (64 and 128 μg/mL) compared with model group (p < 0.05), which showed these compounds inhibit the combination of LPS with receptor of cells; immunohistochemical staining and ELISA method showed the TLR4 receptor expression, IL-6 and TNF-α levels were significant decreased in the groups treated with compounds, indicating that geniposides, baicalin, palmatine and berberine can play the role of anti-sepsis by inhibiting the expression of TLR4, the releasing of IL-6 and TNF-α; MTT assay showed that palmatine and berberine had a weak effect on cell viability, while others not, indicating that the compounds have protective activity. It could be concluded the high affinity binding between these compounds and lipid A may be an important basis for its anti-LPS activity in vitro.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

ECL-IAA and ECL-GADA Can Identify High-Risk Single Autoantibody-Positive Relatives in the TrialNet Pathway to Prevention Study.

PubMed

Steck, Andrea K; Fouts, Alexandra; Miao, Dongmei; Zhao, Zhiyuan; Dong, Fran; Sosenko, Jay; Gottlieb, Peter; Rewers, Marian J; Yu, Liping

2016-07-01

Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies. TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits. After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time. Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects.

High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

NASA Astrophysics Data System (ADS)

Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

2012-03-01

We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

The Cosmos greenstone succession, Agnew-Wiluna greenstone belt, Yilgarn Craton, Western Australia: Geochemistry of an enriched Neoarchaean volcanic arc succession

NASA Astrophysics Data System (ADS)

de Joux, A.; Thordarson, T.; Fitton, J. G.; Hastie, A. R.

2014-09-01

The geodynamic setting of the Neoarchaean Eastern Goldfields Superterrane (EGS) of the Yilgarn Craton is the subject of debate. Some authors propose plume models, while others advocate variants on a subduction accretion model for the origin of mineralised greenstone belt sequences. Felsic volcanism in the Kalgoorlie Terrane, the westernmost terrane of the EGS, is considered to have a tonalite-trondhjemite-granodiorite/dacite (TTG/D) geochemical affinity. The Cosmos greenstone succession, which lies in the Agnew-Wiluna greenstone belt (AWB) of the Kalgoorlie Terrane, contains several komatiite-hosted nickel sulphide deposits, the volcanic footwall to which consists of an intercalated succession of fragmental and coherent rocks ranging in composition from basaltic andesite to rhyolite. Light rare earth elements (LREEs) and large ion-lithophile elements (LILEs) are strongly enriched relative to high field strength elements (HFSEs) across all volcanic units, and the rocks display strong positive Pb and negative Nb anomalies. These geochemical characteristics resemble closely those of modern high-K calc-alkaline to shoshonite continental arc successions. Contrasting REE, LILE and HFSE concentrations, coupled with assimilation-fractional crystallisation (AFC) modelling, shows that the intercalated dacitic and andesitic volcanic rocks within the footwall succession are not co-genetic. Xenocrystic zircons within the felsic volcanic lithologies indicate that some assimilation of older continental crust contributed to the generation of the footwall volcanic sequence. The geochemical characteristics of the Cosmos volcanic succession indicate that parental melts were derived via partial melting of enriched peridotite that had been contaminated by subducted crustal material within the mantle wedge of a subduction zone. In contrast, two younger felsic porphyry intrusions, which cross-cut the volcanic succession, have a distinct TTG/D affinity. Therefore, these intrusions are considered to be generated via partial melting of a subducting slab and are related to local high-Ca granitoid intrusions. The Cosmos volcanic succession represents the first extrusive high-K calc-alkaline to shoshonitic volcanic arc sequence described in the Kalgoorlie Terrane and, coupled with age dating of the stratigraphy, is indicative of formation in a long-lived volcanic arc setting active from 2736 Ma to later than 2724 Ma. The composition and geochemical affinity of the volcanic footwall succession to the Cosmos komatiite-hosted nickel-sulphide deposits contrasts with the majority of felsic volcanic rocks within the AWB and also the wider Kalgoorlie Terrane, suggesting that the overall architecture of this region is more complex than is currently thought. Our conclusions not only have consequences for recent models of the tectonic evolution of the EGS but also contribute to the debate on the operation of plate tectonics during the late Archaean in general. The arc affinity of the Cosmos volcanic succession, containing abundant high-K calc-alkaline andesite lavas, provides further support for the operation of plate tectonics in the Neoarchaean.

High-throughput purification of recombinant proteins using self-cleaving intein tags.

PubMed

Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

2017-01-01

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

Fast probing of glucose and fructose in plant tissues via plasmonic affinity sandwich assay with molecularly-imprinted extraction microprobes.

PubMed

Muhammad, Pir; Liu, Jia; Xing, Rongrong; Wen, Yanrong; Wang, Yijia; Liu, Zhen

2017-12-01

Determination of specific target compounds in agriculture food and natural plant products is essential for many purposes; however, it is often challenging due to the complexity of the sample matrices. Herein we present a new approach called plasmonic affinity sandwich assay for the facile and rapid probing of glucose and fructose in plant tissues. The approach mainly relies on molecularly imprinted plasmonic extraction microprobes, which were prepared on gold-coated acupuncture needles via boronate affinity controllable oriented surface imprinting with the target monosaccharide as the template molecules. An extraction microprobe was inserted into plant tissues under investigation, which allowed for the specific extraction of glucose or fructose from the tissues. The glucose or fructose molecules extracted on the microprobe were labeled with boronic acid-functionalized Raman-active silver nanoparticles, and thus affinity sandwich complexes were formed on the microprobes. After excess Raman nanotags were washed away, the microprobe was subjected to Raman detection. Upon being irradiated with a laser beam, surface plasmon on the gold-coated microprobes was generated, which further produced plasmon-enhanced Raman scattering of the silver-based nanotags and thereby provided sensitive detection. Apple fruits, which contain abundant glucose and fructose, were used as a model of plant tissues. The approach exhibited high specificity, good sensitivity (limit of detection, 1 μg mL -1 ), and fast speed (the whole procedure required only 20 min). The spatial distribution profiles of glucose and fructose within an apple were investigated by the developed approach. Copyright © 2017 Elsevier B.V. All rights reserved.

Lithological architecture and petrography of the Mako Birimian greenstone belt, Kédougou-Kéniéba Inlier, eastern Senegal

NASA Astrophysics Data System (ADS)

Dabo, Moussa; Aïfa, Tahar; Gning, Ibrahima; Faye, Malick; Ba, Mamadou Fallou; Ngom, Papa Malick

2017-07-01

The new lithological and petrographic data obtained in the Mako sector are analyzed in the light of the geochemical data available in the literature. It consists of ultramaic, mafic rocks of tholeiitic affinities associated with intermediate and felsic rocks of calc-alkaline affinities and with intercalations of sedimentary rocks. The whole unit is intruded by Eburnean granitoids and affected by a greenschist to amphibolite facies metamorphism related to a high grade hydrothermalism. It consists of: (i) ultramafic rocks composed of a fractional crystallization succession of lherzolites, wehrlites and pyroxenites with mafic rock inclusions; (ii) layered, isotropic and pegmatitic metagabbros which gradually pass to metabasalts occur at the top; (iii) massive and in pillow metabasalts with locally tapered vesicles, completely or partially filled with quartzo-feldspathic minerals; (iv) quarzites locally overlying the mafic rocks and thus forming the top of the lower unit. This ultramafic-mafic lower unit presents a tholeiitic affinity near to the OIB or N-MORB. It represents the Mako Ophiolitic Complex (MOC), a lithospheric fragment of Birimian lithospheric crust. The upper unit is a mixed volcanic complex arranged in the tectonic corridors. From bottom to top it comprises the following: (i) andesitic, and (ii) rhyodacitic and rhyolitic lava flows and tuffs, respectively. They present a calc-alkaline affinity of the active margins. Three generations of Eburnean granitoids are recognized: (i) early (2215-2160 Ma); (ii) syn-tectonics (2150-2100 Ma) and post-tectonics (2090-2040 Ma). The lithological succession, geochemical and metamorphic characteristics of these units point to an ophiolitic supra-subduction zone.

Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP).

PubMed

Guan, Dongli; Chen, Zhilei

2017-01-01

Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn 2+ ) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use.

Deep sequencing methods for protein engineering and design.

PubMed

Wrenbeck, Emily E; Faber, Matthew S; Whitehead, Timothy A

2017-08-01

The advent of next-generation sequencing (NGS) has revolutionized protein science, and the development of complementary methods enabling NGS-driven protein engineering have followed. In general, these experiments address the functional consequences of thousands of protein variants in a massively parallel manner using genotype-phenotype linked high-throughput functional screens followed by DNA counting via deep sequencing. We highlight the use of information rich datasets to engineer protein molecular recognition. Examples include the creation of multiple dual-affinity Fabs targeting structurally dissimilar epitopes and engineering of a broad germline-targeted anti-HIV-1 immunogen. Additionally, we highlight the generation of enzyme fitness landscapes for conducting fundamental studies of protein behavior and evolution. We conclude with discussion of technological advances. Copyright © 2016 Elsevier Ltd. All rights reserved.

Opposing intermolecular tuning of Ca2+ affinity for Calmodulin by its target peptides

NASA Astrophysics Data System (ADS)

Cheung, Margaret

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with non-equilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM binds to the CaMKII peptide with higher affinity than the Ng peptide. The binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide, which binds to Ca2+-free CaM or Ca2+/CaM with similar binding affinity. Unlike CaM-CaMKII peptide that allowed structure determination by crystallography, the structural description of CaM-Ng peptide is unknown due to low binding affinity, therefore, we computationally generated an ensemble of CaM-Ng peptide structures by matching the changes in the chemical shifts of CaM upon Ng peptide binding from nuclear magnetic resonance experiments. We computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+release. In contrast, CaMKII increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops.

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

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

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

DOE PAGES

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.; ...

2018-02-14

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

PubMed Central

2018-01-01

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutations increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed. PMID:29442996

Design Method of Digital Optimal Control Scheme and Multiple Paralleled Bridge Type Current Amplifier for Generating Gradient Magnetic Fields in MRI Systems

NASA Astrophysics Data System (ADS)

Watanabe, Shuji; Takano, Hiroshi; Fukuda, Hiroya; Hiraki, Eiji; Nakaoka, Mutsuo

This paper deals with a digital control scheme of multiple paralleled high frequency switching current amplifier with four-quadrant chopper for generating gradient magnetic fields in MRI (Magnetic Resonance Imaging) systems. In order to track high precise current pattern in Gradient Coils (GC), the proposal current amplifier cancels the switching current ripples in GC with each other and designed optimum switching gate pulse patterns without influences of the large filter current ripple amplitude. The optimal control implementation and the linear control theory in GC current amplifiers have affinity to each other with excellent characteristics. The digital control system can be realized easily through the digital control implementation, DSPs or microprocessors. Multiple-parallel operational microprocessors realize two or higher paralleled GC current pattern tracking amplifier with optimal control design and excellent results are given for improving the image quality of MRI systems.

Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

PubMed

Lim, Theam Soon; Chan, Soo Khim

2016-01-01

Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

PubMed

Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

2016-08-26

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

Predicting the dynamics of bacterial growth inhibition by ribosome-targeting antibiotics

NASA Astrophysics Data System (ADS)

Greulich, Philip; Doležal, Jakub; Scott, Matthew; Evans, Martin R.; Allen, Rosalind J.

2017-12-01

Understanding how antibiotics inhibit bacteria can help to reduce antibiotic use and hence avoid antimicrobial resistance—yet few theoretical models exist for bacterial growth inhibition by a clinically relevant antibiotic treatment regimen. In particular, in the clinic, antibiotic treatment is time-dependent. Here, we use a theoretical model, previously applied to steady-state bacterial growth, to predict the dynamical response of a bacterial cell to a time-dependent dose of ribosome-targeting antibiotic. Our results depend strongly on whether the antibiotic shows reversible transport and/or low-affinity ribosome binding (‘low-affinity antibiotic’) or, in contrast, irreversible transport and/or high affinity ribosome binding (‘high-affinity antibiotic’). For low-affinity antibiotics, our model predicts that growth inhibition depends on the duration of the antibiotic pulse, and can show a transient period of very fast growth following removal of the antibiotic. For high-affinity antibiotics, growth inhibition depends on peak dosage rather than dose duration, and the model predicts a pronounced post-antibiotic effect, due to hysteresis, in which growth can be suppressed for long times after the antibiotic dose has ended. These predictions are experimentally testable and may be of clinical significance.

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

Beaumont, K.; Vaughn, D.A.; Fanestil, D.D.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. The authors report here that ({sup 3}H)metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. ({sup 3}H)Metolazone bound with high affinity to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or not binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated withmore » their clinical potency. Halide anions specifically inhibited high-affinity binding of ({sup 3}H)metolazone to this site. ({sup 3})Metolazone also bound with lower affinity to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had K{sub i} values of 0.6-10 {mu}M for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity ({sup 3}H)metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.« less

Predicting the dynamics of bacterial growth inhibition by ribosome-targeting antibiotics

PubMed Central

Greulich, Philip; Doležal, Jakub; Scott, Matthew; Evans, Martin R; Allen, Rosalind J

2017-01-01

Understanding how antibiotics inhibit bacteria can help to reduce antibiotic use and hence avoid antimicrobial resistance—yet few theoretical models exist for bacterial growth inhibition by a clinically relevant antibiotic treatment regimen. In particular, in the clinic, antibiotic treatment is time-dependent. Here, we use a theoretical model, previously applied to steady-state bacterial growth, to predict the dynamical response of a bacterial cell to a time-dependent dose of ribosome-targeting antibiotic. Our results depend strongly on whether the antibiotic shows reversible transport and/or low-affinity ribosome binding (‘low-affinity antibiotic’) or, in contrast, irreversible transport and/or high affinity ribosome binding (‘high-affinity antibiotic’). For low-affinity antibiotics, our model predicts that growth inhibition depends on the duration of the antibiotic pulse, and can show a transient period of very fast growth following removal of the antibiotic. For high-affinity antibiotics, growth inhibition depends on peak dosage rather than dose duration, and the model predicts a pronounced post-antibiotic effect, due to hysteresis, in which growth can be suppressed for long times after the antibiotic dose has ended. These predictions are experimentally testable and may be of clinical significance. PMID:28714461

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

PubMed

Suzuki, N; Mihashi, K

1991-01-01

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

PubMed

Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

1993-12-01

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

Cholera Toxin Inhibitors Studied with High-Performance Liquid Affinity Chromatography: A Robust Method to Evaluate Receptor–Ligand Interactions

PubMed Central

Bergström, Maria; Liu, Shuang; Kiick, Kristi L.; Ohlson, Sten

2009-01-01

Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The KD values of galactose and meta-nitrophenyl α-D-galactoside were determined with weak affinity chromatography to be 52 and 1 mM, respectively, which agree well with IC50 values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but KD values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct KD values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis. PMID:19152642

Selection of imprinted nanoparticles by affinity chromatography.

PubMed

Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

2009-04-15

Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

Canine Comfort: Pet Affinity Buffers the Negative Impact of Ambivalence over Emotional Expression on Perceived Social Support.

PubMed

Bryan, Jennifer L; Quist, Michelle C; Young, Chelsie M; Steers, Mai-Ly N; Foster, Dawn W; Lu, Qian

2014-10-01

This study evaluated pet affinity as a buffer between ambivalence over emotional expression (AEE) and social support. AEE occurs when one desires to express emotions but is reluctant to do so and is related to negative psychological outcomes. Individuals high in AEE may have difficulty receiving social support and thus may not gain accompanying benefits. Social support has been associated with positive health outcomes, and pet support is positively associated with human social support. The present study explores the potential protective effect of pet affinity. One hundred ninety-eight undergraduate dog owners completed measures assessing perceived social support, pet affinity, and AEE. AEE was expected to be negatively associated with social support, and pet affinity was expected to buffer the negative effects of AEE on social support. We found that AEE was negatively associated with perceived social support. An interaction between pet affinity and AEE emerged such that the negative association between AEE and social support was weaker among those higher in pet affinity. Thus, at high levels of AEE, those who felt a close connection with their pets reported more perceived social support than those less connected with their pets. Overall, these findings emphasize the potential benefits of pet affinity.

Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

PubMed Central

Arur, Swathi; Schedl, Tim

2014-01-01

Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

Plasma cell output from germinal centers is regulated by signals from Tfh and stromal cells

PubMed Central

George, Laura A.; Acs, Andreas; Durrett, Russell E.

2018-01-01

Germinal centers (GCs) are the sites where B cells undergo affinity maturation. The regulation of cellular output from the GC is not well understood. Here, we show that from the earliest stages of the GC response, plasmablasts emerge at the GC–T zone interface (GTI). We define two main factors that regulate this process: Tfh-derived IL-21, which supports production of plasmablasts from the GC, and TNFSF13 (APRIL), which is produced by a population of podoplanin+ CD157high fibroblastic reticular cells located in the GTI that are also rich in message for IL-6 and chemokines CXCL12, CCL19, and CCL21. Plasmablasts in the GTI express the APRIL receptor TNFRSF13B (TACI), and blocking TACI interactions specifically reduces the numbers of plasmablasts appearing in the GTI. Plasma cells generated in the GTI may provide an early source of affinity-matured antibodies that may neutralize pathogens or provide feedback regulating GC B cell selection. PMID:29549115

Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

PubMed

Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E; Levesque, Brié; Pedersen, Stine B; Bartels, Lina; Wapenaar, Hannah; Ye, Fei; Zhang, Mingjie; Bowen, Mark E; Strømgaard, Kristian

2017-09-15

The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

Application of histone modification-specific interaction domains as an alternative to antibodies.

PubMed

Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul; Jeltsch, Albert

2014-11-01

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. © 2014 Kungulovski et al.; Published by Cold Spring Harbor Laboratory Press.

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

PubMed

Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

2014-06-01

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

PubMed

White, Jim F; Grisshammer, Reinhard

2010-09-07

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

PubMed Central

Ehrlicher, Allen J.; Krishnan, Ramaswamy; Guo, Ming; Bidan, Cécile M.; Weitz, David A.; Pollak, Martin R.

2015-01-01

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease. PMID:25918384

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

A Targeted "Capture" and "Removal" Scavenger toward Multiple Pollutants for Water Remediation based on Molecular Recognition.

PubMed

Wang, Jie; Shen, Haijing; Hu, Xiaoxia; Li, Yan; Li, Zhihao; Xu, Jinfan; Song, Xiufeng; Zeng, Haibo; Yuan, Quan

2016-03-01

For the water remediation techniques based on adsorption, the long-standing contradictories between selectivity and multiple adsorbability, as well as between affinity and recyclability, have put it on weak defense amid more and more severe environment crisis. Here, a pollutant-targeting hydrogel scavenger is reported for water remediation with both high selectivity and multiple adsorbability for several pollutants, and with strong affinity and good recyclability through rationally integrating the advantages of multiple functional materials. In the scavenger, aptamers fold into binding pockets to accommodate the molecular structure of pollutants to afford perfect selectivity, and Janus nanoparticles with antibacterial function as well as anisotropic surfaces to immobilize multiple aptamers allow for simultaneously handling different kinds of pollutants. The scavenger exhibits high efficiencies in removing pollutants from water and it can be easily recycled for many times without significant loss of loading capacities. Moreover, the residual concentrations of each contaminant are well below the drinking water standards. Thermodynamic behavior of the adsorption process is investigated and the rate-controlling process is determined. Furthermore, a point of use device is constructed and it displays high efficiency in removing pollutants from environmental water. The scavenger exhibits great promise to be applied in the next generation of water purification systems.

Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPIα: Difference between FV1 and FV2.

PubMed

van Doorn, Peter; Rosing, Jan; Duckers, Connie; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta

2018-06-04

 Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPIα), presumably by promoting TFPIα binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype.  This article demonstrates the TFPIα-cofactor function of FV in plasma and compares FV1 and FV2.  Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPIα-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPIα-mediated FXa inhibition.  In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPIα antibodies, suggesting that it reflects TFPIα-cofactor activity of FV. In the model system of TFPIα-mediated FXa inhibition, FV2 was a more potent TFPIα-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors.  FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF. Schattauer GmbH Stuttgart.

Direct Identification of On-Bead Peptides Using Surface-Enhanced Raman Spectroscopic Barcoding System for High-Throughput Bioanalysis

PubMed Central

Kang, Homan; Jeong, Sinyoung; Koh, Yul; Geun Cha, Myeong; Yang, Jin-Kyoung; Kyeong, San; Kim, Jaehi; Kwak, Seon-Yeong; Chang, Hye-Jin; Lee, Hyunmi; Jeong, Cheolhwan; Kim, Jong-Ho; Jun, Bong-Hyun; Kim, Yong-Kweon; Hong Jeong, Dae; Lee, Yoon-Sik

2015-01-01

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery. PMID:26017924

Direct identification of on-bead peptides using surface-enhanced Raman spectroscopic barcoding system for high-throughput bioanalysis.

PubMed

Kang, Homan; Jeong, Sinyoung; Koh, Yul; Geun Cha, Myeong; Yang, Jin-Kyoung; Kyeong, San; Kim, Jaehi; Kwak, Seon-Yeong; Chang, Hye-Jin; Lee, Hyunmi; Jeong, Cheolhwan; Kim, Jong-Ho; Jun, Bong-Hyun; Kim, Yong-Kweon; Hong Jeong, Dae; Lee, Yoon-Sik

2015-05-28

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.

Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein.

PubMed

Siqueira, Andrei Santos; Lima, Alex Ranieri Jerônimo; Dall'Agnol, Leonardo Teixeira; de Azevedo, Juliana Simão Nina; da Silva Gonçalves Vianez, João Lídio; Gonçalves, Evonnildo Costa

2016-03-01

Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme-substrate complexation of Cyanobium sp. CACIAM14 showed values around -10 kcal mol(-1) using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco's affinity.

Pointwise regularity of parameterized affine zipper fractal curves

NASA Astrophysics Data System (ADS)

Bárány, Balázs; Kiss, Gergely; Kolossváry, István

2018-05-01

We study the pointwise regularity of zipper fractal curves generated by affine mappings. Under the assumption of dominated splitting of index-1, we calculate the Hausdorff dimension of the level sets of the pointwise Hölder exponent for a subinterval of the spectrum. We give an equivalent characterization for the existence of regular pointwise Hölder exponent for Lebesgue almost every point. In this case, we extend the multifractal analysis to the full spectrum. In particular, we apply our results for de Rham’s curve.

The M sub 1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

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

Mei, Lin.

1989-01-01

The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated ({sup 3}H)IP{sub 1} accumulation in the SH-SY5Y cells was decreased in the presence of 1{mu}g/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M{sub 1} mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m{sub 1} gene. The transfected B82 cells (cTB10) showed specific ({supmore » 3}H)(-)QNB binding activity. The mAChRs in these cells are of the M{sub 1} type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M{sub 1} mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M{sub 1} mAChR densities in these cells characterized by ({sup 3}H)(-)MQNB binding ranged from 12 fmol/10{sup 6} cells in LK3-1 cells to 260 fmol/10{sup 6} cells in the LK3-8 cells.« less

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

PubMed

Shi, Mei; Bennett, Teresa A; Cimino, Daniel F; Maestas, Diane C; Foutz, Terry D; Gurevich, Vsevolod V; Sklar, Larry A; Prossnitz, Eric R

2003-06-24

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

PubMed

Hu, Zu-Quan; Li, He-Ping; Wu, Ping; Li, Ya-Bo; Zhou, Zhu-Qing; Zhang, Jing-Bo; Liu, Jin-Long; Liao, Yu-Cai

2015-03-31

Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2.

PubMed

Song, Jie; Baker, Nicola; Rothert, Monja; Henke, Björn; Jeacock, Laura; Horn, David; Beitz, Eric

2016-02-01

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.

Thermodynamic basis for engineering high-affinity, high-specificity binding-induced DNA clamp nanoswitches.

PubMed

Idili, Andrea; Plaxco, Kevin W; Vallée-Bélisle, Alexis; Ricci, Francesco

2013-12-23

Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson-Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe's affinity for its target by ∼0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide an additional specificity check that increases the discrimination efficiency toward a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the "specificity window" of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.

A new efficient method of generating photoaffinity beads for drug target identification.

PubMed

Nishiya, Yoichi; Hamada, Tomoko; Abe, Masayuki; Takashima, Michio; Tsutsumi, Kyoko; Okawa, Katsuya

2017-02-15

Affinity purification is one of the most prevalent methods for the target identification of small molecules. Preparation of an appropriate chemical for immobilization, however, is a tedious and time-consuming process. A decade ago, a photoreaction method for generating affinity beads was reported, where compounds are mixed with agarose beads carrying a photoreactive group (aryldiazirine) and then irradiated with ultraviolet light under dry conditions to form covalent attachment. Although the method has proven useful for identifying drug targets, the beads suffer from inefficient ligand incorporation and tend to shrink and aggregate, which can cause nonspecific binding and low reproducibility. We therefore decided to craft affinity beads free from these shortcomings without compromising the ease of preparation. We herein report a modified method; first, a compound of interest is mixed with a crosslinker having an activated ester and a photoreactive moiety on each end. This mixture is then dried in a glass tube and irradiated with ultraviolet light. Finally, the conjugates are dissolved and reacted with agarose beads with a primary amine. This protocol enabled us to immobilize compounds more efficiently (approximately 500-fold per bead compared to the original method) and generated beads without physical deterioration. We herein demonstrated that the new FK506-immobilized beads specifically isolated more FKBP12 than the original beads, thereby proving our method to be applicable to target identification experiments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthetic single domain antibodies for the conformational trapping of membrane proteins

PubMed Central

Arnold, Fabian M; Stohler, Peter; Bocquet, Nicolas; Hug, Melanie N; Huber, Sylwia; Siegrist, Martin; Hetemann, Lisa; Gera, Jennifer; Gmür, Samira; Spies, Peter; Gygax, Daniel

2018-01-01

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins. PMID:29792401

Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications.

PubMed

Disney, Alexander J M; Kellam, Barrie; Dekker, Lodewijk V

2016-05-06

The natural product staurosporine is a high-affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4'-methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein-staurosporine conjugate binds to cAMP-dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Engineered Knottin Peptides: A New Class of Agents for Imaging Integrin Expression in Living Subjects

PubMed Central

Kimura, Richard H; Cheng, Zhen; Gambhir, Sanjiv Sam; Cochran, Jennifer R

2009-01-01

There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with low nM affinity to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or 64Cu-DOTA to their N-termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. Near-infrared fluorescence and microPET imaging both demonstrated that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 h post injection for two high affinity (IC50 ∼20 nM) 64Cu-DOTA-conjugated knottin peptides was 4.47 ± 1.21 and 4.56 ± 0.64 % injected dose/gram (%ID/g), compared to a low affinity knottin peptide (IC50 ∼0.4 μM; 1.48 ± 0.53 %ID/g) and c(RGDyK) (IC50 ∼1 μM; 2.32 ± 0.55 %ID/g), a low affinity cyclic pentapeptide under clinical development. Furthermore, 64Cu-DOTA-conjugated knottin peptides generated lower levels of non-specific liver uptake (∼2 %ID/g) compared to c(RGDyK) (∼4 %ID/g) 1 h post injection. MicroPET imaging results were confirmed by in vivo biodistribution studies. 64Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers. PMID:19276378

Selectivity of the uptake of glutamate and GABA in two morphologically distinct insect neuromuscular synapses.

PubMed

van Marle, J; Piek, T; Lammertse, T; Lind, A; Van Weeren-Kramer, J

1985-11-25

The common inhibitor (CI) and slow excitor tibiae (SETi) innervated slow muscles 135cd of the locust Schistocerca gregaria were incubated under high-affinity uptake conditions either in [3H]GABA or in [3H]glutamate. [3H]GABA is accumulated in the glia of the nerve endings of the CI as well as the SETi; however, it is accumulated only in the terminal axons of the CI, not in the terminal axons of the SETi. The grain densities above the glia and above the CI terminal axons are approximately 2 grains/micron2. After incubation in [3H]glutamate the grain densities above the CI terminal axons and the SETi terminal axons are approximately 4 grains/micron2; the grain densities above the glia of both types of nerve endings are approximately 17 grains/micron2. The relatively high labeling (3 grains/micron2) of the muscles after incubation in the presence of glutamate is ascribed to the high metabolic requirements of slow muscles. The conclusion is drawn that a high-affinity uptake system for GABA is present in the CI terminal axons and in the glia of both the CI and SETi nerve endings. However, while the glutamate uptake in the CI and SETi nerve endings of the slow 135cd is comparable to the high-affinity uptake of glutamate in the fast excitor tibiae (FETi) nerve endings of the fast retractor unguis muscle, a high-affinity uptake of glutamate was only demonstrated in the glia of both types of nerve endings. A high-affinity uptake in the terminal axons of the CI and SETi may be masked by an extensively low-affinity uptake of glutamate by the muscles.

Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide.

PubMed

Heiat, Mohammad; Ranjbar, Reza; Latifi, Ali Mohammad; Rasaee, Mohammad Javad

2016-08-01

Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52±2.44E-10 and 5.87±1.3E-9M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33±1.15E-9 and 4.11±1.09E-9M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P<0.05). They also significantly reduced the serum sodium level and increased the urine volume (P<0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity. Copyright © 2016 Elsevier Inc. All rights reserved.

ECL-IAA and ECL-GADA Can Identify High-Risk Single Autoantibody-Positive Relatives in the TrialNet Pathway to Prevention Study

PubMed Central

Fouts, Alexandra; Miao, Dongmei; Zhao, Zhiyuan; Dong, Fran; Sosenko, Jay; Gottlieb, Peter; Rewers, Marian J.

2016-01-01

Abstract Background: Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies. Materials and Methods: TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits. Results: After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time. Conclusions: Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects. PMID:26991969

Isolation and purification of wheat germ agglutinin and analysis of its properties

NASA Astrophysics Data System (ADS)

Wang, Han

2017-12-01

In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

PubMed

Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

Pharmacological characterization of the cloned kappa opioid receptor as a kappa 1b subtype.

PubMed

Lai, J; Ma, S W; Zhu, R H; Rothman, R B; Lentes, K U; Porreca, F

1994-10-27

Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.

Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

PubMed

Sioud, Mouldy; Westby, Phuong; Vasovic, Vlada; Fløisand, Yngvar; Peng, Qian

2018-04-16

mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

The protein-protein interface evolution acts in a similar way to antibody affinity maturation.

PubMed

Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

2010-02-05

Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.

How Structure Defines Affinity in Protein-Protein Interactions

PubMed Central

Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

2014-01-01

Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

PubMed

Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

2014-02-01

Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

PubMed Central

Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela AE; Krauss, Jürgen

2014-01-01

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential. PMID:24256717

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential.

PubMed

Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela A E; Krauss, Jürgen

2014-01-01

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

Immunoglobulin gene usage in the human anti-pathogen response.

PubMed

Newkirk, M M; Rioux, J D

1995-09-01

The human antibody response to foreign pathogens is generated to a relatively small number of target surface proteins and carbohydrates that nonetheless have an extensive array of epitopes. The study of human monoclonal antibodies to different pathogens shows that there are a diversity of mechanisms used to generate a sufficient repertoire of antibodies to combat the invading pathogens. Although many different immunoglobulin gene elements are used to construct the anti-pathogen response, some elements are used more often than would be expected if all elements were used randomly. For example, the immune response to Haemophilus influenzae polysaccharide appears to be quite narrow, being restricted primarily to a specific heavy-chain gene, 3-15, and a lambda light-chain family II member, 4A. In contrast, for the immune response to cytomegalovirus proteins, a wider group of gene elements is needed. It is also surprising that despite an investigator bias for IgG- rather than IgM-secreting immortal B cells (because of their high affinity and neutralizing abilities), 26% of light chains and 13% of heavy chains showed a very low level of somatic mutation, equivalent to an IgM molecule that has not undergone affinity maturation. Although some highly mutated IgG molecules are present in the anti-pathogen response, most of the monoclonal antibodies specific for viruses or bacteria have a level of somatic hypermutation similar to that of the adult IgM repertoire. A number of studies have shown that there are similarities in the antibody responses to pathogens and to self (autoantibodies).(ABSTRACT TRUNCATED AT 250 WORDS)

Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.

PubMed

Prakash, Thazha P; Graham, Mark J; Yu, Jinghua; Carty, Rick; Low, Audrey; Chappell, Alfred; Schmidt, Karsten; Zhao, Chenguang; Aghajan, Mariam; Murray, Heather F; Riney, Stan; Booten, Sheri L; Murray, Susan F; Gaus, Hans; Crosby, Jeff; Lima, Walt F; Guo, Shuling; Monia, Brett P; Swayze, Eric E; Seth, Punit P

2014-07-01

Triantennary N-acetyl galactosamine (GalNAc, GN3: ), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6-10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2'-O-Et-2',4'-bridged nucleic acid) gapmer ASOs, ∼ 60-fold enhancement in potency relative to the parent MOE (2'-O-methoxyethyl RNA) ASO was observed. GN3: -conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3: -ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3: -ASO conjugate was detected in plasma suggesting that GN3: acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications.

PubMed

Shetty, Dinesh; Khedkar, Jayshree K; Park, Kyeng Min; Kim, Kimoon

2015-12-07

The design of synthetic, monovalent host-guest molecular recognition pairs is still challenging and of particular interest to inquire into the limits of the affinity that can be achieved with designed systems. In this regard, cucurbit[7]uril (CB[7]), an important member of the host family cucurbit[n]uril (CB[n], n = 5-8, 10, 14), has attracted much attention because of its ability to form ultra-stable complexes with multiple guests. The strong hydrophobic effect between the host cavity and guests, ion-dipole and dipole-dipole interactions of guests with CB portals helps in cooperative and multiple noncovalent interactions that are essential for realizing such strong complexations. These highly selective, strong yet dynamic interactions can be exploited in many applications including affinity chromatography, biomolecule immobilization, protein isolation, biological catalysis, and sensor technologies. In this review, we summarize the progress in the development of high affinity guests for CB[7], factors affecting the stability of complexes, theoretical insights, and the utility of these high affinity pairs in different challenging applications.

A 45-Amino-Acid Scaffold Mined from the PDB for High-Affinity Ligand Engineering.

PubMed

Kruziki, Max A; Bhatnagar, Sumit; Woldring, Daniel R; Duong, Vandon T; Hackel, Benjamin J

2015-07-23

Small protein ligands can provide superior physiological distribution compared with antibodies, and improved stability, production, and specific conjugation. Systematic evaluation of the PDB identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α helix opposite a β sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 10(8) mutants and directed evolution toward four targets yielded target-specific binders with affinities as strong as 200 ± 100 pM, Tms from 65 °C ± 3 °C to 80°C ± 1 °C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ± 8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold. Copyright © 2015 Elsevier Ltd. All rights reserved.

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

Mobile Technology Affinity in Renal Transplant Recipients.

PubMed

Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

PubMed

Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

2017-01-01

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

PubMed

Kanomata, Naoki; Matsuura, Shiro; Nomura, Tsunehisa; Kurebayashi, Junichi; Mori, Taisuke; Kitawaki, Jo; Moriya, Takuya

2017-01-01

Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between aromatase expression and aromatase inhibitors are warranted.

Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

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

Whitehead, Timothy A.; Chevalier, Aaron; Song, Yifan

2012-06-19

We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followedmore » by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.« less

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

PubMed

Lundegaard, Claus; Lamberth, Kasper; Harndahl, Mikkel; Buus, Søren; Lund, Ole; Nielsen, Morten

2008-07-01

NetMHC-3.0 is trained on a large number of quantitative peptide data using both affinity data from the Immune Epitope Database and Analysis Resource (IEDB) and elution data from SYFPEITHI. The method generates high-accuracy predictions of major histocompatibility complex (MHC): peptide binding. The predictions are based on artificial neural networks trained on data from 55 MHC alleles (43 Human and 12 non-human), and position-specific scoring matrices (PSSMs) for additional 67 HLA alleles. As only the MHC class I prediction server is available, predictions are possible for peptides of length 8-11 for all 122 alleles. artificial neural network predictions are given as actual IC(50) values whereas PSSM predictions are given as a log-odds likelihood scores. The output is optionally available as download for easy post-processing. The training method underlying the server is the best available, and has been used to predict possible MHC-binding peptides in a series of pathogen viral proteomes including SARS, Influenza and HIV, resulting in an average of 75-80% confirmed MHC binders. Here, the performance is further validated and benchmarked using a large set of newly published affinity data, non-redundant to the training set. The server is free of use and available at: http://www.cbs.dtu.dk/services/NetMHC.

A twice-as-smart synthetic G-quartet: PyroTASQ is both a smart quadruplex ligand and a smart fluorescent probe.

PubMed

Laguerre, Aurélien; Stefan, Loic; Larrouy, Manuel; Genest, David; Novotna, Jana; Pirrotta, Marc; Monchaud, David

2014-09-03

Recent and unambiguous evidences of the formation of DNA and RNA G-quadruplexes in cells has provided solid support for these structures to be considered as valuable targets in oncology. Beyond this, they have lent further credence to the anticancer strategies relying on small molecules that selectively target these higher-order DNA/RNA architectures, referred to as G-quadruplex ligands. They have also shed bright light on the necessity of designing multitasking ligands, displaying not only enticing quadruplex interacting properties (affinity, structural selectivity) but also additional features that make them usable for detecting quadruplexes in living cells, notably for determining whether, when, and where these structures fold and unfold during the cell cycle and also for better assessing the consequences of their stabilization by external agents. Herein, we report a brand new design of such multitasking ligands, whose structure experiences a quadruplex-promoted conformational switch that triggers not only its quadruplex affinity (i.e., smart ligands, which display high affinity and selectivity for DNA/RNA quadruplexes) but also its fluorescence (i.e., smart probes, which behave as selective light-up fluorescent reporters on the basis of a fluorogenic electron redistribution). The first prototype of such multifunctional ligands, termed PyroTASQ, represents a brand new generation of quadruplex ligands that can be referred to as "twice-as-smart" quadruplex ligands.

Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.

PubMed

Huang, Renhua; Fang, Pete; Kay, Brian K

2012-09-01

Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. Copyright © 2012 Elsevier Inc. All rights reserved.

Identification and Cloning of Centaurin-α

PubMed Central

Hammonds-Odie, Latanya P.; Jackson, Trevor R.; Profit, Adam A.; Blader, Ira J.; Turck, Christoph W.; Prestwich, Glenn D.; Theibert, Anne B.

2015-01-01

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-α. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-α displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nm), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-α is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain. PMID:8702546

The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6

PubMed Central

Boschert, V.; Frisch, C.; Back, J. W.; van Pee, K.; Weidauer, S. E.; Muth, E.-M.; Schmieder, P.; Beerbaum, M.; Knappik, A.; Timmerman, P.

2016-01-01

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis. PMID:27558933

Role of hydrogen bonding in ligand interaction with the N-methyl-D-aspartate receptor ion channel

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

Leeson, P.D.; Carling, R.W.; James, K.

1990-05-01

Displacement of (3H)MK-801 (dizocilpine, 1) binding to rat brain membranes has been used to evaluate the affinities of novel dibenzocycloalkenimines related to 1 for the ion channel binding site (also known as the phencyclidine or PCP receptor) on the N-methyl-D-aspartate (NMDA) subtype of excitory amino acid receptor. In common with many other agents having actions in the central nervous system, these compounds contain a hydrophobic aromatic moiety and a basic nitrogen atom. The conformational rigidity of these ligands provides a unique opportunity to evaluate the importance of specific geometrical properties that influence active-site recognition, in particular the role of themore » nitrogen atom in hydrogen-bonding interactions. The relative affinities (IC50s) of hydrocarbon-substituted analogues of 1 and ring homologated cyclooctenimines illustrate the importance of size-limited hydrophobic binding of both aryl rings and of the quaternary C-5 methyl group. Analysis of the binding of a series of the 10 available structurally rigid dibenzoazabicyclo(x.y.z)alkanes, by using molecular modeling techniques, uncovered a highly significant correlation between affinity and a proposed ligand-active site hydrogen bonding vector (r = 0.950, p less than 0.001). These results are used to generate a pharmacophore of the MK-801 recognition site/PCP receptor, which accounts for the binding of all of the known ligands.« less

SFG characterization of a cationic ONLO dye in biological thin films

NASA Astrophysics Data System (ADS)

Johnson, Lewis E.; Casford, Michael T.; Elder, Delwin L.; Davies, Paul B.; Johal, Malkiat S.

2013-10-01

Biopolymer-based thin films, such as those composed of CTMA-DNA, can be used as a host material for NLOactive dyes for applications such as electro-optic (EO) switching and second harmonic generation. Previous work by Heckman et al. (Proc. SPIE 6401, 640108-2) has demonstrated functioning DNA-based EO modulators. Improved performance requires optimization of both the first hyperpolarizabilities (β) and degree of acentric ordering exhibited by the chromophores. The cationic dye DANPY-1 (Proc. SPIE 8464, 846409-D) has a high affinity for DNA and a substantial hyperpolarizability; however, its macroscopic ordering has not been previously characterized. We have characterized the acentric ordering of the dye using sum-frequency generation (SFG) vibrational spectroscopy in surface-immobilized DNA and on planar metal and dielectric surfaces.

Novel near-infrared spectrum analysis tool: Synergy adaptive moving window model based on immune clone algorithm.

PubMed

Wang, Shenghao; Zhang, Yuyan; Cao, Fuyi; Pei, Zhenying; Gao, Xuewei; Zhang, Xu; Zhao, Yong

2018-02-13

This paper presents a novel spectrum analysis tool named synergy adaptive moving window modeling based on immune clone algorithm (SA-MWM-ICA) considering the tedious and inconvenient labor involved in the selection of pre-processing methods and spectral variables by prior experience. In this work, immune clone algorithm is first introduced into the spectrum analysis field as a new optimization strategy, covering the shortage of the relative traditional methods. Based on the working principle of the human immune system, the performance of the quantitative model is regarded as antigen, and a special vector corresponding to the above mentioned antigen is regarded as antibody. The antibody contains a pre-processing method optimization region which is created by 11 decimal digits, and a spectrum variable optimization region which is formed by some moving windows with changeable width and position. A set of original antibodies are created by modeling with this algorithm. After calculating the affinity of these antibodies, those with high affinity will be selected to clone. The regulation for cloning is that the higher the affinity, the more copies will be. In the next step, another import operation named hyper-mutation is applied to the antibodies after cloning. Moreover, the regulation for hyper-mutation is that the lower the affinity, the more possibility will be. Several antibodies with high affinity will be created on the basis of these steps. Groups of simulated dataset, gasoline near-infrared spectra dataset, and soil near-infrared spectra dataset are employed to verify and illustrate the performance of SA-MWM-ICA. Analysis results show that the performance of the quantitative models adopted by SA-MWM-ICA are better especially for structures with relatively complex spectra than traditional models such as partial least squares (PLS), moving window PLS (MWPLS), genetic algorithm PLS (GAPLS), and pretreatment method classification and adjustable parameter changeable size moving window PLS (CA-CSMWPLS). The selected pre-processing methods and spectrum variables are easily explained. The proposed method will converge in few generations and can be used not only for near-infrared spectroscopy analysis but also for other similar spectral analysis, such as infrared spectroscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Zukin, R.S.; Eghbali, M.; Olive, D.

{kappa} opioid receptors ({kappa} receptors) have been characterized in homogenates of guinea pig and rat brain under in vitro binding conditions. {kappa} receptors were labeled by using the tritiated prototypic {kappa} opioid ethylketocyclazocine under conditions in which {mu} and {delta} opioid binding was suppressed. In the case of guinea pig brain membranes, a single population of high-affinity {kappa} opioid receptor sites was observed. In contrast, in the case of rat brain, two populations of {kappa} sites were observed. To test the hypothesis that the high- and low-affinity {kappa} sites represent two distinct {kappa} receptor subtypes, a series of opioids weremore » tested for their abilities to compete for binding to the two sites. U-69,593 and Cambridge 20 selectively displaced the high-affinity {kappa} site in both guinea pig and rat tissue, but were inactive at the rat-brain low-affinity site. Other {kappa} opioid drugs competed for binding to both sites, but with different rank orders of potency. Quantitative light microscopy in vitro autoradiography was used to visualize the neuroanatomical pattern of {kappa} receptors in rat and guinea pig brain. The distribution patterns of the two {kappa} receptor subtypes of rat brain were clearly different. Collectively, these data provide direct evidence for the presence of two {kappa} receptor subtypes; the U-69,593-sensitive, high-affinity {kappa}{sub 1} site predominates in guinea pig brain, and the U-69,593-insensitive, low-affinity {kappa}{sub 2} site predominates in rat brain.« less

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis

PubMed Central

Pillon, Arnaud; Boussioux, Anne-Marie; Escande, Aurélie; Aït-Aïssa, Sélim; Gomez, Elena; Fenet, Hélène; Ruff, Marc; Moras, Dino; Vignon, Françoise; Duchesne, Marie-Josèphe; Casellas, Claude; Nicolas, Jean-Claude; Balaguer, Patrick

2005-01-01

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis. PMID:15743715

[Specific effects of Cr ions on DNA: a comparison between the interaction of CrIII with purified DNA and with DNA from cultured cells].

PubMed

Balbi, C; Vecchio, D; Russo, P; Parodi, S; Santi, L

1981-05-30

Affinity between CrIII and purified calf thymus DNA were studied by equilibrium dialysis at different pHs. Chromium was dosed by atomic spectrometry. This affinity was compared with Chromium-DNA affinity after treatment of living mammalian cells with CrVI and its intracellular reduction. Preliminary results seem to suggest that affinity is similar in both cases and not especially high (K approximately 10(5) 1/mole).

Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells.

PubMed

Miyanokoshi, Miki; Yokosawa, Takumi; Wakasugi, Keisuke

2018-06-01

The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon-γ (IFN-γ)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN-γ-treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN-γ-treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II).

PubMed

Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin

2017-06-16

Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC 50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

PubMed

Lai, H; Carino, M A

1992-07-01

Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

The FOXP2 forkhead domain binds to a variety of DNA sequences with different rates and affinities.

PubMed

Webb, Helen; Steeb, Olga; Blane, Ashleigh; Rotherham, Lia; Aron, Shaun; Machanick, Philip; Dirr, Heini; Fanucchi, Sylvia

2017-07-01

FOXP2 is a member of the P subfamily of FOX transcription factors, the DNA-binding domain of which is the winged helix forkhead domain (FHD). In this work we show that the FOXP2 FHD is able to bind to various DNA sequences, including a novel sequence identified in this work, with different affinities and rates as detected using surface plasmon resonance. Combining the experimental work with molecular docking, we show that high-affinity sequences remain bound to the protein for longer, form a greater number of interactions with the protein and induce a greater structural change in the protein than low-affinity sequences. We propose a binding model for the FOXP2 FHD that involves three types of binding sequence: low affinity sites which allow for rapid scanning of the genome by the protein in a partially unstructured state; moderate affinity sites which serve to locate the protein near target sites and high-affinity sites which secure the protein to the DNA and induce a conformational change necessary for functional binding and the possible initiation of downstream transcriptional events. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Imprinting the Fate of Antigen-Reactive B Cells through the Affinity of the B Cell Receptor

PubMed Central

O'Connor, Brian P.; Vogel, Laura A.; Zhang, Weijun; Loo, William; Shnider, Danielle; Lind, Evan F.; Ratliff, Michelle; Noelle, Randolph J.; Erickson, Loren D.

2010-01-01

Long-lived plasma cells (PCs) and memory B cells (Bmem) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short-or long-lived PCs and Bmem. Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither Bmem nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but Bmem remain extinct. In contrast, lower affinity interactions show tempered GCs, producing Bmem and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity. PMID:17114443

Dimeric MHC-peptides inserted into an immunoglobulin scaffold as new immunotherapeutic agents

PubMed Central

Goldberg, Burt; Bona, Constantin

2011-01-01

Abstract The interactions of the T cell receptor (TCR) with cognate MHC-peptide and co-stimulatory molecules expressed at surface of antigen presenting cells (APC) leads to activation or tolerance of T cells. The development of molecular biological tools allowed for the preparation of soluble MHC-peptide molecules as surrogate for the APC. A decade ago a monomeric class II MHC molecule in which the peptide was covalently linked to β-chain of class II molecule was generated. This type of molecule had a low-binding affinity and did not cause the multimerization of TCR. The requirement of multimerization of TCR led to development of a new class of reagents, chimeric peptides covalently linked to MHC that was dimerized via Fc fragment of an immunoglobulin and linked to 3′ end of the β-chain of MHC class II molecule. These soluble dimerized MHC-peptide chimeric molecules display high affinity for the TCR and caused multimerization of TCR without processing by an APC. Because dimeric molecules are devoid of co-stimulatory molecules interacting with CD28, a second signal, they induce anergy rather the activation of T cells. In this review, we compare the human and murine dimerized MHC class II-peptides and their effect on CD4+ T cells, particularly the generation of T regulatory cells, which make these chimeric molecules an appealing approach for the treatment of autoimmune diseases. PMID:21435177

Preparation and characterization of fluorophenylboronic acid-functionalized affinity monolithic columns for the selective enrichment of cis-diol-containing biomolecules.

PubMed

Li, Qianjin; Liu, Zhen

2015-01-01

Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity monolithic column applying 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) as a ligand. The DFFPBA-functionalized monolithic column not only exhibited an ultrahigh boronate affinity toward cis-diol-containing biomolecules, but also showed great potential for the selective enrichment of cis-diol-containing biomolecules in real samples.

Evaluation of Computer Tools for Idea Generation and Team Formation in Project-Based Learning

ERIC Educational Resources Information Center

Ardaiz-Villanueva, Oscar; Nicuesa-Chacon, Xabier; Brene-Artazcoz, Oscar; Sanz de Acedo Lizarraga, Maria Luisa; Sanz de Acedo Baquedano, Maria Teresa

2011-01-01

The main objective of this research was to validate the effectiveness of Wikideas and Creativity Connector tools to stimulate the generation of ideas and originality by university students organized into groups according to their indexes of creativity and affinity. Another goal of the study was to evaluate the classroom climate created by these…

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

PubMed

Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

2016-01-01

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva.

PubMed

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui; Xiao, Yi

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

The localization and concentration of the PDE2-encoded high-affinity cAMP phosphodiesterase is regulated by cAMP-dependent protein kinase A in the yeast Saccharomyces cerevisiae.

PubMed

Hu, Yun; Liu, Enkai; Bai, Xiaojia; Zhang, Aili

2010-03-01

The genome of the yeast Saccharomyces cerevisiae encodes two cyclic AMP (cAMP) phosphodiesterases, a low-affinity one, Pde1, and a high-affinity one, Pde2. Pde1 has been ascribed a function for downregulating agonist-induced cAMP accumulation in a protein kinase A (PKA)-governed negative feedback loop, whereas Pde2 controls the basal cAMP level in the cell. Here we show that PKA regulates the localization and protein concentration of Pde2. Pde2 is accumulated in the nucleus in wild-type cells growing on glucose, or in strains with hyperactive PKA. In contrast, in derepressed wild-type cells or cells with attenuated PKA activity, Pde2 is distributed over the nucleus and cytoplasm. We also show evidence indicating that the Pde2 protein level is positively correlated with PKA activity. The increase in the Pde2 protein level in high-PKA strains and in cells growing on glucose was due to its increased half-life. These results suggest that, like its low-affinity counterpart, the high-affinity phosphodiesterase may also play an important role in the PKA-controlled feedback inhibition of intracellular cAMP.

Activated tumor cell integrin αvβ3 cooperates with platelets to promote extravasation and metastasis from the blood stream.

PubMed

Weber, Martin R; Zuka, Masahiko; Lorger, Mihaela; Tschan, Mario; Torbett, Bruce E; Zijlstra, Andries; Quigley, James P; Staflin, Karin; Eliceiri, Brian P; Krueger, Joseph S; Marchese, Patrizia; Ruggeri, Zaverio M; Felding, Brunhilde H

2016-04-01

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease. © 2016 Elsevier Ltd. All rights reserved.

Activated tumor cell integrin αvβ3 cooperates with platelets to promote extravasation and metastasis from the blood stream

PubMed Central

Weber, Martin R.; Zuka, Masahiko; Lorger, Mihaela; Tschan, Mario; Torbett, Bruce E.; Zijlstra, Andries; Quigley, James P.; Staflin, Karin; Eliceiri, Brian P.; Krueger, Joseph S.; Marchese, Patricia; Ruggeri, Zaverio M.; Felding, Brunhilde H.

2016-01-01

Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease. PMID:27067975

Computational design of an endo-1,4-[beta]-xylanase ligand binding site

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

Morin, Andrew; Kaufmann, Kristian W.; Fortenberry, Carie

2012-09-05

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-{beta}-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterizationmore » of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 {beta}-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the {beta}-xylanase proteins.« less

Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

PubMed

Lips, Katrin S; Pfeil, Uwe; Reiners, Katja; Rimasch, Christoph; Kuchelmeister, Klaus; Braun-Dullaeus, Ruediger C; Haberberger, Rainer V; Schmidt, Rupert; Kummer, Wolfgang

2003-12-01

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.

Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

PubMed Central

Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K.; Arora, Karunesh; Brooks, Charles L.; Raghavan, Malini

2015-01-01

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin–substrate interactions and as key determinants of PLC dynamics. PMID:26420867

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

PubMed

Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

2013-12-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity

PubMed Central

Sharma, P.; Postel, S.; Sundberg, E.J.; Kranz, D.M.

2013-01-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

MCL-CAw: a refinement of MCL for detecting yeast complexes from weighted PPI networks by incorporating core-attachment structure

PubMed Central

2010-01-01

Background The reconstruction of protein complexes from the physical interactome of organisms serves as a building block towards understanding the higher level organization of the cell. Over the past few years, several independent high-throughput experiments have helped to catalogue enormous amount of physical protein interaction data from organisms such as yeast. However, these individual datasets show lack of correlation with each other and also contain substantial number of false positives (noise). Over these years, several affinity scoring schemes have also been devised to improve the qualities of these datasets. Therefore, the challenge now is to detect meaningful as well as novel complexes from protein interaction (PPI) networks derived by combining datasets from multiple sources and by making use of these affinity scoring schemes. In the attempt towards tackling this challenge, the Markov Clustering algorithm (MCL) has proved to be a popular and reasonably successful method, mainly due to its scalability, robustness, and ability to work on scored (weighted) networks. However, MCL produces many noisy clusters, which either do not match known complexes or have additional proteins that reduce the accuracies of correctly predicted complexes. Results Inspired by recent experimental observations by Gavin and colleagues on the modularity structure in yeast complexes and the distinctive properties of "core" and "attachment" proteins, we develop a core-attachment based refinement method coupled to MCL for reconstruction of yeast complexes from scored (weighted) PPI networks. We combine physical interactions from two recent "pull-down" experiments to generate an unscored PPI network. We then score this network using available affinity scoring schemes to generate multiple scored PPI networks. The evaluation of our method (called MCL-CAw) on these networks shows that: (i) MCL-CAw derives larger number of yeast complexes and with better accuracies than MCL, particularly in the presence of natural noise; (ii) Affinity scoring can effectively reduce the impact of noise on MCL-CAw and thereby improve the quality (precision and recall) of its predicted complexes; (iii) MCL-CAw responds well to most available scoring schemes. We discuss several instances where MCL-CAw was successful in deriving meaningful complexes, and where it missed a few proteins or whole complexes due to affinity scoring of the networks. We compare MCL-CAw with several recent complex detection algorithms on unscored and scored networks, and assess the relative performance of the algorithms on these networks. Further, we study the impact of augmenting physical datasets with computationally inferred interactions for complex detection. Finally, we analyse the essentiality of proteins within predicted complexes to understand a possible correlation between protein essentiality and their ability to form complexes. Conclusions We demonstrate that core-attachment based refinement in MCL-CAw improves the predictions of MCL on yeast PPI networks. We show that affinity scoring improves the performance of MCL-CAw. PMID:20939868

Alternating carrier models of asymmetric glucose transport violate the energy conservation laws.

PubMed

Naftalin, Richard J

2008-11-01

Alternating access transporters with high-affinity externally facing sites and low-affinity internal sites relate substrate transit directly to the unliganded asymmetric "carrier" (Ci) distribution. When both bathing solutions contain equimolar concentrations of ligand, zero net flow of the substrate-carrier complex requires a higher proportion of unliganded low-affinity inside sites (proportional, variant 1/KD(in)) and slower unliganded "free" carrier transit from inside to outside than in the reverse direction. However, asymmetric rates of unliganded carrier movement, kij, imply that an energy source, DeltaGcarrier = RT ln (koi/kio) = RT ln (Cin/Cout) = RT ln (KD(in)/KD(out)), where R is the universal gas constant (8.314 Joules/M/K degrees), and T is the temperature, assumed here to be 300 K degrees , sustains the asymmetry. Without this invalid assumption, the constraints of carrier path cyclicity, combined with asymmetric ligand affinities and equimolarity at equilibrium, are irreconcilable, and any passive asymmetric uniporter or cotransporter model system, e.g., Na-glucose cotransporters, espousing this fundamental error is untenable. With glucose transport via GLUT1, the higher maximal rate and Km of net ligand exit compared to net ligand entry is only properly simulated if ligand transit occurs by serial dissociation-association reactions between external high-affinity and internal low-affinity immobile sites. Faster intersite transit rates occur from lower-affinity sites than from higher-affinity sites and require no other energy source to maintain equilibrium. Similar constraints must apply to cotransport.

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Robust feature matching via support-line voting and affine-invariant ratios

NASA Astrophysics Data System (ADS)

Li, Jiayuan; Hu, Qingwu; Ai, Mingyao; Zhong, Ruofei

2017-10-01

Robust image matching is crucial for many applications of remote sensing and photogrammetry, such as image fusion, image registration, and change detection. In this paper, we propose a robust feature matching method based on support-line voting and affine-invariant ratios. We first use popular feature matching algorithms, such as SIFT, to obtain a set of initial matches. A support-line descriptor based on multiple adaptive binning gradient histograms is subsequently applied in the support-line voting stage to filter outliers. In addition, we use affine-invariant ratios computed by a two-line structure to refine the matching results and estimate the local affine transformation. The local affine model is more robust to distortions caused by elevation differences than the global affine transformation, especially for high-resolution remote sensing images and UAV images. Thus, the proposed method is suitable for both rigid and non-rigid image matching problems. Finally, we extract as many high-precision correspondences as possible based on the local affine extension and build a grid-wise affine model for remote sensing image registration. We compare the proposed method with six state-of-the-art algorithms on several data sets and show that our method significantly outperforms the other methods. The proposed method achieves 94.46% average precision on 15 challenging remote sensing image pairs, while the second-best method, RANSAC, only achieves 70.3%. In addition, the number of detected correct matches of the proposed method is approximately four times the number of initial SIFT matches.

HIV protease drug resistance and its impact on inhibitor design.

PubMed

Ala, P J; Rodgers, J D; Chang, C H

1999-07-01

The primary cause of resistance to the currently available HIV protease inhibitors is the accumulation of multiple mutations in the viral protease. So far more than 20 substitutions have been observed in the active site, dimer interface, surface loops and flaps of the homodimer. While many mutations reduce the protease's affinity for inhibitors, others appear to enhance its catalytic efficiency. This high degree of genetic flexibility has made the protease an elusive drug target. The design of the next generation of HIV protease inhibitors will be discussed in light of the current structural information.

Generation of a pair of independently binding DNA aptamers in a single round of selection using proximity ligation.

PubMed

Chumphukam, O; Le, T T; Piletsky, S; Cass, A E G

2015-05-28

The ability to rapidly generate a pair of aptamers that bind independently to a protein target would greatly extend their use as reagents for two site ('sandwich') assays. We describe here a method to achieve this through proximity ligation. Using lysozyme as a target we demonstrate that under optimal conditions such a pair of aptamers, with nanomolar affinities, can be generated in a single round.

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

PubMed

Tossavainen, Helena; Aitio, Olli; Hellman, Maarit; Saksela, Kalle; Permi, Perttu

2016-07-29

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications

PubMed Central

Hage, David S.

2017-01-01

BACKGROUND The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically-related binding agent, are two methods that can be used to study these interactions. CONTENT This review looks at various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent. SUMMARY The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples. PMID:28396561

Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

PubMed

Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

2018-04-25

Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Affinity ranking of antibodies using flow cytometry: application in antibody phage display-based target discovery.

PubMed

Geuijen, Cecilia A W; Clijsters-van der Horst, Marieke; Cox, Freek; Rood, Pauline M L; Throsby, Mark; Jongeneelen, Mandy A C; Backus, Harold H J; van Deventer, Els; Kruisbeek, Ada M; Goudsmit, Jaap; de Kruif, John

2005-07-01

Application of antibody phage display to the identification of cell surface antigens with restricted expression patterns is often complicated by the inability to demonstrate specific binding to a certain cell type. The specificity of an antibody can only be properly assessed when the antibody is of sufficient high affinity to detect low-density antigens on cell surfaces. Therefore, a robust and simple assay for the prediction of relative antibody affinities was developed and compared to data obtained using surface plasmon resonance (SPR) technology. A panel of eight anti-CD46 antibody fragments with different affinities was selected from phage display libraries and reformatted into complete human IgG1 molecules. SPR was used to determine K(D) values for these antibodies. The association and dissociation of the antibodies for binding to CD46 expressed on cell surfaces were analysed using FACS-based assays. We show that ranking of the antibodies based on FACS data correlates well with ranking based on K(D) values as measured by SPR and can therefore be used to discriminate between high- and low-affinity antibodies. Finally, we show that a low-affinity antibody may only detect high expression levels of a surface marker while failing to detect lower expression levels of this molecule, which may lead to a false interpretation of antibody specificity.

The binding properties of cycloxaprid on insect native nAChRs partially explain the low cross-resistance with imidacloprid in Nilaparvata lugens.

PubMed

Zhang, Yixi; Xu, Xiaoyong; Bao, Haibo; Shao, Xusheng; Li, Zhong; Liu, Zewen

2018-06-06

Neonicotinoids, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) to control Nilaparvata lugens, a major rice insect pest. High imidacloprid resistance has been reported in N. lugens in laboratory and in fields. Cycloxaprid, an oxabridged cis-nitromethylene neonicotinoid, showed high insecticidal activity against N. lugens and low cross-resistance in the imidacloprid resistant strains and field populations. Binding studies have demonstrated that imidacloprid had two binding sites with different affinities (Kd = 3.18 ± 0.43 pM and 1.78 ± 0.19 nM) in N. lugens nAChRs. Cycloxaprid was poor at displacing [ 3 H]imidacloprid at its high-affinity binding site (Ki = 159.38±20.43 nM), but quite efficient at the low-affinity binding site (Ki = 1.27±0.35 nM). These data showed that cycloxaprid had overlapping binding sites with imidacloprid only at its low-affinity binding site. Therefore, the low displacement ability of cycloxaprid against imidacloprid binding at its high affinity site could partially explain the low cross-resistance of cycloxaprid in the imidacloprid resistant populations. The high insecticidal activity, low cross-resistance and different binding properties on insect nAChRs of cycloxaprid demonstrating it a potential insecticide to control N. lugens and related insect pests, especially the ones with high resistance to neonicotinoids. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The Phytopathogen Pseudomonas syringae pv. tomato DC3000 Has Three High-Affinity Iron-Scavenging Systems Functional under Iron Limitation Conditions but Dispensable for Pathogenesis▿¶

PubMed Central

Jones, Alexander M.; Wildermuth, Mary C.

2011-01-01

High-affinity iron scavenging through the use of siderophores is a well-established virulence determinant in mammalian pathogenesis. However, few examples have been reported for plant pathogens. Here, we use a genetic approach to investigate the role of siderophores in Pseudomonas syringae pv. tomato DC3000 (DC3000) virulence in tomato. DC3000, an agronomically important pathogen, has two known siderophores for high-affinity iron scavenging, yersiniabactin and pyoverdin, and we uncover a third siderophore, citrate, required for growth when iron is limiting. Though growth of a DC3000 triple mutant unable to either synthesize or import these siderophores is severely restricted in iron-limited culture, it is fully pathogenic. One explanation for this phenotype is that the DC3000 triple mutant is able to directly pirate plant iron compounds such as heme/hemin or iron-nicotianamine, and our data indicate that DC3000 can import iron-nicotianamine with high affinity. However, an alternative explanation, supported by data from others, is that the pathogenic environment of DC3000 (i.e., leaf apoplast) is not iron limited but is iron replete, with available iron of >1 μM. Growth of the triple mutant in culture is restored to wild-type levels by supplementation with a variety of iron chelates at >1 μM, including iron(III) dicitrate, a dominant chelate of the leaf apoplast. This suggests that lower-affinity iron import would be sufficient for DC3000 iron nutrition in planta and is in sharp contrast to the high-affinity iron-scavenging mechanisms required in mammalian pathogenesis. PMID:21441525

Immunotherapy expands and maintains the function of high affinity tumor infiltrating CD8 T cells in situ

PubMed Central

Moran, Amy E.; Polesso, Fanny; Weinberg, Andrew D.

2016-01-01

Cancer cells harbor high affinity tumor-associated antigens capable of eliciting potent anti-tumor T cell responses yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and PD-1 have been used. We report here that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor-antigen specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Co-expression of Nur77GFP and OX40 identifies a polyclonal population of high affinity tumor-associated antigen-specific CD8+ T cells, which produce more IFNγ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or PD-L1. Moreover, expansion of these high affinity CD8 T cells prolongs survival of tumor bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor resident CD8 T cells thereby increasing the frequency of cytolytic, high affinity, tumor-associated antigen-specific cells. PMID:27503208

SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

EPA Science Inventory

Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

EPA Science Inventory

Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

Are cannabidiol and Δ(9) -tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review.

PubMed

McPartland, John M; Duncan, Marnie; Di Marzo, Vincenzo; Pertwee, Roger G

2015-02-01

Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ(9) -tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ(9) -tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target. © 2014 The British Pharmacological Society.

Are cannabidiol and Δ9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review

PubMed Central

McPartland, John M; Duncan, Marnie; Di Marzo, Vincenzo; Pertwee, Roger G

2015-01-01

Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ9-tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ9-tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target. PMID:25257544

Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies

PubMed Central

Hwang, Joyce K.; Wang, Chong; Du, Zhou; Meyers, Robin M.; Kepler, Thomas B.; Neuberg, Donna; Kwong, Peter D.; Mascola, John R.; Joyce, M. Gordon; Bonsignori, Mattia; Haynes, Barton F.; Yeap, Leng-Siew; Alt, Frederick W.

2017-01-01

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1–infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms. PMID:28747530

Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter.

PubMed Central

Zelcer, Noam; Saeki, Tohru; Bot, Ilse; Kuil, Annemieke; Borst, Piet

2003-01-01

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect ( Spodoptera frugiperda ) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC50 approximately 10 microM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50 approximately 100, 250 and 500 microM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a K(m) and V(max) of 29+/-7 microM and 660 +/- 63 pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis. PMID:12220224

A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

PubMed

Molero, Anabel; Vendrell, Marc; Bonaventura, Jordi; Zachmann, Julian; López, Laura; Pardo, Leonardo; Lluis, Carme; Cortés, Antoni; Albericio, Fernando; Casadó, Vicent; Royo, Miriam

2015-06-05

Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

PubMed Central

Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

2006-01-01

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

PubMed Central

Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

2016-01-01

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

IA-2 autoantibody affinity in children at risk for type 1 diabetes.

PubMed

Krause, Stephanie; Chmiel, Ruth; Bonifacio, Ezio; Scholz, Marlon; Powell, Michael; Furmaniak, Jadwiga; Rees Smith, Bernard; Ziegler, Anette-G; Achenbach, Peter

2012-12-01

Autoantibodies to insulinoma-associated protein 2 (IA-2A) are associated with increased risk for type 1 diabetes. Here we examined IA-2A affinity and epitope specificity to assess heterogeneity in response intensity in relation to pathogenesis and diabetes risk in 50 children who were prospectively followed from birth. At first IA-2A appearance, affinity ranged from 10(7) to 10(11)L/mol and was high (>1.0×10(9)L/mol) in 41 (82%) children. IA-2A affinity was not associated with epitope specificity or HLA class II haplotype. On follow-up, affinity increased or remained high, and IA-2A were commonly against epitopes within the protein tyrosine phosphatase-like IA-2 domain and the homologue protein IA-2β. IA-2A were preceded or accompanied by other islet autoantibodies in 49 (98%) children, of which 34 progressed to diabetes. IA-2A affinity did not stratify diabetes risk. In conclusion, the IA-2A response in children is intense with rapid maturation against immunogenic epitopes and a strong association with diabetes development. Copyright © 2012 Elsevier Inc. All rights reserved.

Solubilization and purification of melatonin receptors from lizard brain

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

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency

PubMed Central

Walter, Jolan E.; Rucci, Francesca; Patrizi, Laura; Recher, Mike; Regenass, Stephan; Paganini, Tiziana; Keszei, Marton; Pessach, Itai; Lang, Philipp A.; Poliani, Pietro Luigi; Giliani, Silvia; Al-Herz, Waleed; Cowan, Morton J.; Puck, Jennifer M.; Bleesing, Jack; Niehues, Tim; Schuetz, Catharina; Malech, Harry; DeRavin, Suk See; Facchetti, Fabio; Gennery, Andrew R.; Andersson, Emma; Kamani, Naynesh R.; Sekiguchi, JoAnn; Alenezi, Hamid M.; Chinen, Javier; Dbaibo, Ghassan; ElGhazali, Gehad; Fontana, Adriano; Pasic, Srdjan; Detre, Cynthia; Terhorst, Cox

2010-01-01

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro–B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP–keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4+ T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell–activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations. PMID:20547827

Conservation laws and symmetries in stochastic thermodynamics.

PubMed

Polettini, Matteo; Bulnes-Cuetara, Gregory; Esposito, Massimiliano

2016-11-01

Phenomenological nonequilibrium thermodynamics describes how fluxes of conserved quantities, such as matter, energy, and charge, flow from outer reservoirs across a system and how they irreversibly degrade from one form to another. Stochastic thermodynamics is formulated in terms of probability fluxes circulating in the system's configuration space. The consistency of the two frameworks is granted by the condition of local detailed balance, which specifies the amount of physical quantities exchanged with the reservoirs during single transitions between configurations. We demonstrate that the topology of the configuration space crucially determines the number of independent thermodynamic affinities (forces) that the reservoirs generate across the system and provides a general algorithm that produces the fundamental affinities and their conjugate currents contributing to the total dissipation, based on the interplay between macroscopic conservations laws for the currents and microscopic symmetries of the affinities.

Myelin-reactive “type B” T cells and T cells specific for low-affinity MHC-binding myelin peptides escape tolerance in HLA-DR transgenic mice

PubMed Central

Kawamura, Kazuyuki; McLaughlin, Katherine A.; Weissert, Robert; Forsthuber, Thomas G.

2009-01-01

Genes of the major histocompatibility complex (MHC) show the strongest genetic association with multiple sclerosis (MS) but the underlying mechanisms have remained unresolved. Here, we asked whether the MS-associated MHC class II molecules, HLA-DRB1*1501, HLA-DRB5*0101, and HLA-DRB1*0401 contribute to autoimmune central nervous system (CNS) demyelination by promoting pathogenic T cell responses to human myelin basic protein (hMBP), using three transgenic (Tg) mouse lines expressing these MHC molecules. Unexpectedly, profound T cell tolerance to the high-affinity MHC-binding hMBP82-100 epitope was observed in all Tg mouse lines. T cell tolerance to hMBP82-100 was abolished upon backcrossing the HLA-DR Tg mice to MBP-deficient mice. In contrast, T cell tolerance was incomplete for low-affinity MHC-binding hMBP epitopes. Furthermore, hMBP82-100-specific “type B” T cells escaped tolerance in HLA-DRB5*0101 Tg mice. Importantly, T cells specific for low-affinity MHC-binding hMBP epitopes and hMBP82-100-specific “type B” T cells were highly encephalitogenic. Collectively, the results show that MS-associated MHC class II molecules are highly efficient at inducing T cell tolerance to high-affinity MHC-binding epitope, whereas autoreactive T cells specific for the low-affinity MHC-binding epitopes and “type B” T cells can escape the induction of T cell tolerance and may promote MS. PMID:18713991

ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

PubMed

Hisabori, T; Yoshida, M; Sakurai, H

1986-09-01

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

A Spatially Detailed Model of Isometric Contraction Based on Competitive Binding of Troponin I Explains Cooperative Interactions between Tropomyosin and Crossbridges

PubMed Central

Land, Sander; Niederer, Steven A.

2015-01-01

Biophysical models of cardiac tension development provide a succinct representation of our understanding of force generation in the heart. The link between protein kinetics and interactions that gives rise to high cooperativity is not yet fully explained from experiments or previous biophysical models. We propose a biophysical ODE-based representation of cross-bridge (XB), tropomyosin and troponin within a contractile regulatory unit (RU) to investigate the mechanisms behind cooperative activation, as well as the role of cooperativity in dynamic tension generation across different species. The model includes cooperative interactions between regulatory units (RU-RU), between crossbridges (XB-XB), as well more complex interactions between crossbridges and regulatory units (XB-RU interactions). For the steady-state force-calcium relationship, our framework predicts that: (1) XB-RU effects are key in shifting the half-activation value of the force-calcium relationship towards lower [Ca2+], but have only small effects on cooperativity. (2) XB-XB effects approximately double the duty ratio of myosin, but do not significantly affect cooperativity. (3) RU-RU effects derived from the long-range action of tropomyosin are a major factor in cooperative activation, with each additional unblocked RU increasing the rate of additional RU’s unblocking. (4) Myosin affinity for short (1–4 RU) unblocked stretches of actin of is very low, and the resulting suppression of force at low [Ca2+] is a major contributor in the biphasic force-calcium relationship. We also reproduce isometric tension development across mouse, rat and human at physiological temperature and pacing rate, and conclude that species differences require only changes in myosin affinity and troponin I/troponin C affinity. Furthermore, we show that the calcium dependence of the rate of tension redevelopment ktr is explained by transient blocking of RU’s by a temporary decrease in XB-RU effects. PMID:26262582

Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification.

PubMed

Fujii, Yuki; Kaneko, Mika K; Ogasawara, Satoshi; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Honma, Ryusuke; Kato, Yukinari

2017-04-01

Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the "RAP tag" system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification.

War of the Credit Cards.

ERIC Educational Resources Information Center

Nicklin, Julie L.

1993-01-01

Increasingly, colleges are offering affinity credit cards with attractive incentives as a marketing tool. Some in academe feel the trend may compromise educational objectives. Institutions may also face taxation on unrelated-business income generated through the cards. (MSE)

Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

PubMed Central

Azimi, Mohammad; Mofrad, Mohammad R. K.

2013-01-01

Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

Detachment of affinity-captured bioparticles by elastic deformation of a macroporous hydrogel

PubMed Central

Dainiak, Maria B.; Kumar, Ashok; Galaev, Igor Yu.; Mattiasson, Bo

2006-01-01

Adsorption of bioparticles to affinity surfaces involves polyvalent interactions, complicating greatly the recovery of the adsorbed material. A unique system for the efficient binding and release of different cells and particles is described. Affinity-bound bioparticles and synthetic particles are detached from the macroporous hydrogel matrix, a so-called cryogel, when the cryogel undergoes elastic deformation. The particle detachment upon elastic deformation is believed to be due to breaking of many of the multipoint attachments between the particles and the affinity matrix and the change in the distance between affinity ligands when the matrix is deformed. However, no release of affinity-bound protein occurred upon elastic deformation. The phenomenon of particle detachment upon elastic deformation is believed to be of a generic nature, because it was demonstrated for a variety of bioparticles of different sizes and for synthetic particles, for different ligand–receptor pairs (IgG–protein A, sugar–ConA, metal ion–chelating ligand), and when the deformation was caused by either external forces (mechanical deformation) or internal forces (the shrinkage of thermosensitive, macroporous hydrogel upon an increase in temperature). The elasticity of cryogel monoliths ensures high recovery of captured cells under mild conditions, with highly retained viability. This property, along with their continuous porous structure makes cryogel monoliths very attractive for applications in affinity cell separation. PMID:16418282

Synthesis and evaluation of aryl-naloxamide opiate analgesics targeting truncated exon 11-associated μ opioid receptor (MOR-1) splice variants.

PubMed

Majumdar, Susruta; Subrath, Joan; Le Rouzic, Valerie; Polikar, Lisa; Burgman, Maxim; Nagakura, Kuni; Ocampo, Julie; Haselton, Nathan; Pasternak, Anna R; Grinnell, Steven; Pan, Ying-Xian; Pasternak, Gavril W

2012-07-26

3-Iodobenzoylnaltrexamide 1 (IBNtxA) is a potent analgesic acting through a novel receptor target that lack many side-effects of traditional opiates composed, in part, of exon 11-associated truncated six transmembrane domain MOR-1 (6TM/E11) splice variants. To better understand the SAR of this drug target, a number of 4,5-epoxymorphinan analogues were synthesized. Results show the importance of a free 3-phenolic group, a phenyl ring at the 6 position, an iodine at the 3'or 4' position of the phenyl ring, and an N-allyl or c-propylmethyl group to maintain high 6TM/E11 affinity and activity. 3-Iodobenzoylnaloxamide 15 (IBNalA) with a N-allyl group displayed lower δ opioid receptor affinity than its naltrexamine analogue, was 10-fold more potent an analgesic than morphine, elicited no respiratory depression or physical dependence, and only limited inhibition of gastrointestinal transit. Thus, the aryl-naloxamide scaffold can generate a potent analgesic acting through the 6TM/E11 sites with advantageous side-effect profile and greater selectivity.

The challenges of modelling antibody repertoire dynamics in HIV infection

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

Luo, Shishi; Perelson, Alan S.

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

The challenges of modelling antibody repertoire dynamics in HIV infection

DOE PAGES

Luo, Shishi; Perelson, Alan S.

2015-07-20

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

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

Quantitative insight into the design of compounds recognized by the L-type amino acid transporter 1 (LAT1).

PubMed

Ylikangas, Henna; Malmioja, Kalle; Peura, Lauri; Gynther, Mikko; Nwachukwu, Emmanuel O; Leppänen, Jukka; Laine, Krista; Rautio, Jarkko; Lahtela-Kakkonen, Maija; Huttunen, Kristiina M; Poso, Antti

2014-12-01

L-Type amino acid transporter 1 (LAT1) is a transmembrane protein expressed abundantly at the blood-brain barrier (BBB), where it ensures the transport of hydrophobic acids from the blood to the brain. Due to its unique substrate specificity and high expression at the BBB, LAT1 is an intriguing target for carrier-mediated transport of drugs into the brain. In this study, a comparative molecular field analysis (CoMFA) model with considerable statistical quality (Q(2) =0.53, R(2) =0.75, Q(2) SE=0.77, R(2) SE=0.57) and good external predictivity (CCC=0.91) was generated. The model was used to guide the synthesis of eight new prodrugs whose affinity for LAT1 was tested by using an in situ rat brain perfusion technique. This resulted in the creation of a novel LAT1 prodrug with L-tryptophan as the promoiety; it also provided a better understanding of the molecular features of LAT1-targeted high-affinity prodrugs, as well as their promoiety and parent drug. The results obtained will be beneficial in the rational design of novel LAT1-binding prodrugs and other compounds that bind to LAT1. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efficient killing of CD22{sup +} tumor cells by a humanized diabody-RNase fusion protein

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

Krauss, Juergen; Arndt, Michaela A.E.; Vu, Bang K.

2005-06-03

We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22{sup +} tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V{sub L}36{sub Leu{yields}}{sub Tyr}) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K{sub D} 0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as solublemore » protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22{sup +} tumor cell lines with high efficacy (IC{sub 50} = 3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.« less

Challenges and opportunities in the purification of recombinant tagged proteins.

PubMed

Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

2014-01-01

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. Copyright © 2013 Elsevier Inc. All rights reserved.

Discovery of Tertiary Sulfonamides as Potent Liver X Receptor Antagonists

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

Zuercher, William J.; Buckholz†, Richard G.; Campobasso, Nino

2010-08-12

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

PubMed

Zuercher, William J; Buckholz, Richard G; Campobasso, Nino; Collins, Jon L; Galardi, Cristin M; Gampe, Robert T; Hyatt, Stephen M; Merrihew, Susan L; Moore, John T; Oplinger, Jeffrey A; Reid, Paul R; Spearing, Paul K; Stanley, Thomas B; Stewart, Eugene L; Willson, Timothy M

2010-04-22

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Use of receptor chimeras to identify small molecules with high affinity for the dynorphin A binding domain of the kappa opioid receptor.

PubMed

Kumar, Virendra; Guo, Deqi; Marella, Michael; Cassel, Joel A; Dehaven, Robert N; Daubert, Jeffrey D; Mansson, Erik

2008-06-15

A series of 2-substituted sulfamoyl arylacetamides of general structure 2 were prepared as potent kappa opioid receptor agonists and the affinities of these compounds for opioid and chimeric receptors were compared with those of dynorphin A. Compounds 2e and 2i were identified as non-peptide small molecules that bound to chimeras 3 and 4 with high affinities similar to dynorphin A, resulting in K(i) values of 1.5 and 1.2 nM and 1.3 and 2.2 nM, respectively.

Cyclic peptide unguisin A is an anion receptor with high affinity for phosphate and pyrophosphate.

PubMed

Daryl Ariawan, A; Webb, James E A; Howe, Ethan N W; Gale, Philip A; Thordarson, Pall; Hunter, Luke

2017-04-05

Unguisin A (1) is a marine-derived, GABA-containing cyclic heptapeptide. The biological function of this flexible macrocycle is obscure. Here we show that compound 1 lacks any detectable activity in antimicrobial growth inhibition assays, a result that runs contrary to a previous report. However, we find that 1 functions as a promiscuous host molecule in a variety of anion-binding interactions, with high affinity particularly for phosphate and pyrophosphate. We also show that a series of rigidified, backbone-fluorinated analogues of 1 displays altered affinity for chloride ions.

A Water‐Soluble Tetraazaperopyrene Dye as Strong G‐Quadruplex DNA Binder

PubMed Central

Hahn, Lena

2016-01-01

Abstract The interactions of the water‐soluble tetraazaperopyrene dye 1 with ct‐DNA, duplex‐[(dAdT)12 ⋅(dAdT)12], duplex‐[(dGdC)12 ⋅(dGdC)12] as well as with two G‐quadruplex‐forming sequences, namely the human telomeric 22AG and the promotor sequence c‐myc, were investigated by means of UV/visible and fluorescence spectroscopy, isothermal titration calorimetry (ITC) and molecular docking studies. Dye 1 exhibits a high affinity for G‐quadruplex structures over duplex DNA structures. Furthermore, the ligand shows promising G‐quadruplex discrimination, with an affinity towards c‐myc of 2×107  m −1 (i.e., K d=50 nm), which is higher than for 22AG (4×106  m −1). The ITC data reveal that compound 1 interacts with c‐myc in a stoichiometric ratio of 1:1 but also indicate the presence of two identical lower affinity secondary binding sites per quadruplex. In 22AG, there are two high affinity binding sites per quadruplex, that is, one on each side, with a further four weaker binding sites. For both quadruplex structures, the high affinity interactions between compound 1 and the quadruplex‐forming nucleic acid structures are weakly endothermic. Molecular docking studies suggest an end‐stacking binding mode for compound 1 interacting with quadruplex structures, and a higher affinity for the parallel conformation of c‐myc than for the mixed‐hybrid conformation of 22AG. In addition, docking studies also suggest that the reduced affinity for duplex DNA structures is due to the non‐viability of an intercalative binding mode. PMID:26997208

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

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

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

PubMed

Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

2002-07-01

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

An innovative and highly drug-tolerant approach for detecting neutralizing antibodies directed to therapeutic antibodies.

PubMed

Sloan, John H; Conway, Richard G; Pottanat, Thomas G; Troutt, Jason S; Higgs, Richard E; Konrad, Robert J; Qian, Yue-Wei

2016-10-01

Immunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody. Excess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies. This highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

PubMed

Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd. All rights reserved.

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy.

PubMed

Congdon, Erin E; Lin, Yan; Rajamohamedsait, Hameetha B; Shamir, Dov B; Krishnaswamy, Senthilkumar; Rajamohamedsait, Wajitha J; Rasool, Suhail; Gonzalez, Veronica; Levenga, Josien; Gu, Jiaping; Hoeffer, Charles; Sigurdsson, Einar M

2016-08-30

A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures. Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer's paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6's efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly. Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

Highly sensitive and unbiased approach for elucidating antibody repertoires

PubMed Central

Lin, Sherry G.; Ba, Zhaoqing; Du, Zhou; Zhang, Yu; Hu, Jiazhi; Alt, Frederick W.

2016-01-01

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. PMID:27354528

An interaction map of small-molecule kinase inhibitors with anaplastic lymphoma kinase (ALK) mutants in ALK-positive non-small cell lung cancer.

PubMed

Ai, Xinghao; Shen, Shengping; Shen, Lan; Lu, Shun

2015-05-01

Human anaplastic lymphoma kinase (ALK) has become a well-established target for the treatment of ALK-positive non-small cell lung cancer (NSCLC). Here, we have profiled seven small-molecule inhibitors, including 2 that are approved drugs, against a panel of clinically relevant mutations in ALK tyrosine kinase (TK) domain, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying inhibitor response to ALK TK mutation. We find that (i) the gatekeeper mutation L1196M causes crizotinib resistance by simultaneously increasing and decreasing the binding affinities of, respectively, ATP and inhibitor to ALK, whereas the secondary mutation C1156Y, which is located far away from the ATP-binding site of ALK TK domain, causes the resistance by inducing marked allosteric effect on the site, (ii) the 2nd and 3rd generation kinase inhibitors exhibit relatively high sensitivity towards ALK mutants as compared to 1st generation inhibitors, (iii) the pan-kinase inhibitor staurosporine is insensitive for most mutations due to its high structural compatibility, and (iv) ATP affinity to ALK is generally reduced upon most clinically relevant mutations. Furthermore, we also identify six novel mutation-inhibitor pairs that are potentially associated with drug resistance. In addition, the G1202R and C1156Y mutations are expected to generally cause resistance for many existing inhibitors, since they can address significant effect on the geometric shape and physicochemical property of ALK active pocket. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

The influence of antibody fragment format on phage display based affinity maturation of IgG

PubMed Central

Steinwand, Miriam; Droste, Patrick; Frenzel, Andrè; Hust, Michael; Dübel, Stefan; Schirrmann, Thomas

2014-01-01

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab. PMID:24262918

How high do ion fluxes go? A re-evaluation of the two-mechanism model of K+ 2 transport in plant roots

USDA-ARS?s Scientific Manuscript database

Potassium acquisition in roots is described by a two-mechanism model, consisting of a saturable, high-affinity transport system (HATS) operating via H+/K+ symport at low (< 1 mM) external [K+] ([K+]ext), and a linear, low-affinity system (LATS) operating via ion channels at high (> 112 mM) [K+]ext. ...

Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge

PubMed Central

Kogot, Joshua M.; Zhang, Yanting; Moore, Stephen J.; Pagano, Paul; Stratis-Cullum, Dimitra N.; Chang-Yen, David; Turewicz, Marek; Pellegrino, Paul M.; de Fusco, Andre; Soh, H. Tom; Stagliano, Nancy E.

2011-01-01

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS. PMID:22140433

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1.

PubMed

Baruffini, Enrico; Goffrini, Paola; Donnini, Claudia; Lodi, Tiziana

2006-12-01

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2011-12-08

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

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

PubMed

Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

2015-09-01

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Image Segmentation, Registration, Compression, and Matching

NASA Technical Reports Server (NTRS)

Yadegar, Jacob; Wei, Hai; Yadegar, Joseph; Ray, Nilanjan; Zabuawala, Sakina

2011-01-01

A novel computational framework was developed of a 2D affine invariant matching exploiting a parameter space. Named as affine invariant parameter space (AIPS), the technique can be applied to many image-processing and computer-vision problems, including image registration, template matching, and object tracking from image sequence. The AIPS is formed by the parameters in an affine combination of a set of feature points in the image plane. In cases where the entire image can be assumed to have undergone a single affine transformation, the new AIPS match metric and matching framework becomes very effective (compared with the state-of-the-art methods at the time of this reporting). No knowledge about scaling or any other transformation parameters need to be known a priori to apply the AIPS framework. An automated suite of software tools has been created to provide accurate image segmentation (for data cleaning) and high-quality 2D image and 3D surface registration (for fusing multi-resolution terrain, image, and map data). These tools are capable of supporting existing GIS toolkits already in the marketplace, and will also be usable in a stand-alone fashion. The toolkit applies novel algorithmic approaches for image segmentation, feature extraction, and registration of 2D imagery and 3D surface data, which supports first-pass, batched, fully automatic feature extraction (for segmentation), and registration. A hierarchical and adaptive approach is taken for achieving automatic feature extraction, segmentation, and registration. Surface registration is the process of aligning two (or more) data sets to a common coordinate system, during which the transformation between their different coordinate systems is determined. Also developed here are a novel, volumetric surface modeling and compression technique that provide both quality-guaranteed mesh surface approximations and compaction of the model sizes by efficiently coding the geometry and connectivity/topology components of the generated models. The highly efficient triangular mesh compression compacts the connectivity information at the rate of 1.5-4 bits per vertex (on average for triangle meshes), while reducing the 3D geometry by 40-50 percent. Finally, taking into consideration the characteristics of 3D terrain data, and using the innovative, regularized binary decomposition mesh modeling, a multistage, pattern-drive modeling, and compression technique has been developed to provide an effective framework for compressing digital elevation model (DEM) surfaces, high-resolution aerial imagery, and other types of NASA data.

Probing ligand recognition of the opioid pan antagonist AT-076 at nociceptin, kappa, mu, and delta opioid receptors through structure-activity relationships.

PubMed

Journigan, V Blair; Polgar, Willma E; Tuan, Edward W; Lu, James; Daga, Pankaj R; Zaveri, Nurulain T

2017-10-16

Few opioid ligands binding to the three classic opioid receptor subtypes, mu, kappa and delta, have high affinity at the fourth opioid receptor, the nociceptin/orphanin FQ receptor (NOP). We recently reported the discovery of AT-076 (1), (R)-7-hydroxy-N-((S)-1-(4-(3-hydroxyphenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, a pan antagonist with nanomolar affinity for all four subtypes. Since AT-076 binds with high affinity at all four subtypes, we conducted a structure-activity relationship (SAR) study to probe ligand recognition features important for pan opioid receptor activity, using chemical modifications of key pharmacophoric groups. SAR analysis of the resulting analogs suggests that for the NOP receptor, the entire AT-076 scaffold is crucial for high binding affinity, but the binding mode is likely different from that of NOP antagonists C-24 and SB-612111 bound in the NOP crystal structure. On the other hand, modifications of the 3-hydroxyphenyl pharmacophore, but not the 7-hydroxy Tic pharmacophore, are better tolerated at kappa and mu receptors and yield very high affinity multifunctional (e.g. 12) or highly selective (e.g. 16) kappa ligands. With the availability of the opioid receptor crystal structures, our SAR analysis of the common chemotype of AT-076 suggests rational approaches to modulate binding selectivity, enabling the design of multifunctional or selective opioid ligands from such scaffolds.

Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

PubMed Central

Labonté, Eric D.; Howles, Philip N.; Granholm, Norman A.; Rojas, Juan C.; Davies, Joanna P.; Ioannou, Yiannis A.; Hui, David Y.

2007-01-01

Recent studies have documented the importance of Niemann Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1−/− mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1−/− mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1−/− mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1. PMID:17442616

Novel antipsychotics activate recombinant human and native rat serotonin 5-HT1A receptors: affinity, efficacy and potential implications for treatment of schizophrenia.

PubMed

Newman-Tancredi, Adrian; Assié, Marie-Bernadette; Leduc, Nathalie; Ormière, Anne-Marie; Danty, Nathalie; Cosi, Cristina

2005-09-01

Serotonin 5-HT1A receptors are promising targets in the management of schizophrenia but little information exists about affinity and efficacy of novel antipsychotics at these sites. We addressed this issue by comparing binding affinity at 5-HT1A receptors with dopamine rD2 receptors, which are important targets for antipsychotic drug action. Agonist efficacy at 5-HT1A receptors was determined for G-protein activation and adenylyl cyclase activity. Whereas haloperidol, thioridazine, risperidone and olanzapine did not interact with 5-HT1A receptors, other antipsychotic agents exhibited agonist properties at these sites. E(max) values (% effect induced by 10 microM of 5-HT) for G-protein activation at rat brain 5-HT1A receptors: sarizotan (66.5), bifeprunox (35.9), SSR181507 (25.8), nemonapride (25.7), ziprasidone (20.6), SLV313 (19), aripiprazole (15), tiospirone (8.9). These data were highly correlated with results obtained at recombinant human 5-HT1A receptors in determinations of G-protein activation and inhibition of forskolin-stimulated adenylyl cyclase. In binding-affinity determinations, the antipsychotics exhibited diverse properties at r5-HT1A receptors: sarizotan (pK(i)=8.65), SLV313 (8.64), SSR181507 (8.53), nemonapride (8.35), ziprasidone (8.30), tiospirone (8.22), aripiprazole (7.42), bifeprunox (7.19) and clozapine (6.31). The affinity ratios of the ligands at 5-HT1A vs. D2 receptors also varied widely: ziprasidone, SSR181507 and SLV313 had similar affinities whereas aripiprazole, nemonapride and bifeprunox were more potent at D2 than 5-HT1A receptors. Taken together, these data indicate that aripiprazole has low efficacy and modest affinity at 5-HT1A receptors, whereas bifeprunox has low affinity but high efficacy. In contrast, SSR181507 has intermediate efficacy but high affinity, and is likely to have more prominent 5-HT1A receptor agonist properties. Thus, the contribution of 5-HT1A receptor activation to the pharmacological profile of action of the antipsychotics will depend on the relative 5-HT1A/D2 affinities and on 5-HT1A agonist efficacy of the drugs.

Low-affinity spermine block mediating outward currents through Kir2.1 and Kir2.2 inward rectifier potassium channels

PubMed Central

Ishihara, Keiko; Yan, Ding-Hong

2007-01-01

The outward component of the strong inward rectifier K+ current (IKir) plays a pivotal role in polarizing the membranes of excitable and non-excitable cells and is regulated by voltage-dependent channel block by internal cations. Using the Kir2.1 channel, we previously showed that a small fraction of the conductance susceptible only to a low-affinity mode of block likely carries a large portion of the outward current. To further examine the relevance of the low-affinity block to outward IKir and to explore its molecular mechanism, we studied the block of the Kir2.1 and Kir2.2 channels by spermine, which is the principal Kir2 channel blocker. Current–voltage relations of outward Kir2.2 currents showed a peak, a plateau and two peaks in the presence of 10, 1 and 0.1 μm spermine, respectively, which was explained by the presence of two conductances that differ in their susceptibility to spermine block. When the current–voltage relations showed one peak, like those of native IKir, outward Kir2.2 currents were mediated mostly by the conductance susceptible to the low-affinity block. They also flowed in a narrower range than the corresponding Kir2.1 currents, because of 3- to 4-fold greater susceptibility to the low-affinity block than in Kir2.1. Reducing external [K+] shifted the voltage dependences of both the high- and low-affinity block of Kir2.1 in parallel with the shift in the reversal potential, confirming the importance of the low-affinity block in mediating outward IKir. When Kir2.1 mutants known to have reduced sensitivity to internal blockers were examined, the D172N mutation in the transmembrane pore region made almost all of the conductance susceptible only to low-affinity block, while the E224G mutation in the cytoplasmic pore region reduced the sensitivity to low-affinity block without markedly altering that to the high-affinity block or the high/low conductance ratio. The effects of these mutations support the hypothesis that Kir2 channels exist in two states having different susceptibilities to internal cationic blockers. PMID:17640933

Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

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

Rothman, R.B.; Reid, A.; Mahboubi, A.

1991-02-01

Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and lowmore » affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.« less

Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Tanomand, Asghar; Akbari, Bahman

2016-11-01

Phage display is a prominent screening technique for development of novel high affinity antibodies against almost any antigen. However, removing false positive clones in screening process remains a challenge. The aim of this study was to develop an efficient and rapid method for isolation of high affinity scFvs by removing NSBs without losing rare specific clones. Therefore, a novel two rounds strategy called invert biopanning was developed for isolating high affinity scFvs against EGFRvIII antigen from human scFv library. The efficiency of invert biopanning method (procedure III) was analyzed by comparing with results of conventional biopanning methods (procedures I and II). According to the results of polyclonal ELISA, the second round of procedure III displayed highest binding affinity against EGFRvIII peptide accompanied by lowest NSB comparing to other two procedures. Several positive clones were identified among output phages of procedure III by monoclonal phage ELISA which displayed high affinity to EGFRvIII antigen. In conclusion, results of our study indicate that invert biopanning is an efficient method for avoiding NSBs and conservation of rare specific clones during screening of a scFv phage library. Novel anti EGFRvIII scFv isolated could be a promising candidate for potential use in treatment of EGFRvIII expressing cancers. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

PubMed Central

Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

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.

IFN-γ regulates CD8+ memory T cell differentiation and survival in response to weak, but not strong, TCR signals.

PubMed

Stoycheva, Diana; Deiser, Katrin; Stärck, Lilian; Nishanth, Gopala; Schlüter, Dirk; Uckert, Wolfgang; Schüler, Thomas

2015-01-15

In response to primary Ag contact, naive mouse CD8(+) T cells undergo clonal expansion and differentiate into effector T cells. After pathogen clearance, most effector T cells die, and only a small number of memory T cell precursors (TMPs) survive to form a pool of long-lived memory T cells (TMs). Although high- and low-affinity CD8(+) T cell clones are recruited into the primary response, the TM pool consists mainly of high-affinity clones. It remains unclear whether the more efficient expansion of high-affinity clones and/or cell-intrinsic processes exclude low-affinity T cells from the TM pool. In this article, we show that the lack of IFN-γR signaling in CD8(+) T cells promotes TM formation in response to weak, but not strong, TCR agonists. The IFN-γ-sensitive accumulation of TMs correlates with reduced mammalian target of rapamycin activation and the accumulation of long-lived CD62L(hi)Bcl-2(hi)Eomes(hi) TMPs. Reconstitution of mammalian target of rapamycin or IFN-γR signaling is sufficient to block this process. Hence, our data suggest that IFN-γR signaling actively blocks the formation of TMPs responding to weak TCR agonists, thereby promoting the accumulation of high-affinity T cells finally dominating the TM pool. Copyright © 2015 by The American Association of Immunologists, Inc.

Influence of N-ethylmaleimide on cholinoceptors and responses in longitudinal muscles from guinea-pig ileum.

PubMed Central

Aronstam, R. S.; Carrier, G. O.

1982-01-01

1 The binding of carbamylcholine to membranes prepared from the longitudinal muscle of guinea-pig ileum was determined from its inhibition of the binding of [3H]-3-quinuclidinyl benzilate. Carbamylcholine binding was resolved into high and low affinity components with apparent dissociation constants of 0.11 +/- 0.02 and 11 +/- 1 microM; 42% of the receptors displayed high affinity carbamylcholine binding. 2 Alkylation of longitudinal muscle membranes with N-ethylmaleimide increased muscarinic receptor affinity for carbamylcholine in a manner consistent with a conversion of low affinity to high affinity receptors. After exposure the muscle membrane fragments to 1 mM N-ethylmaleimide for 20 min at 35 degrees C, carbamylcholine binding was resolved into two components with apparent dissociation constants of 0.11 +/- 0.01 and 9 +/- 2 microM, with 74% of the receptors displaying the higher affinity. 3 Exposure of longitudinal membranes mounted in an organ chamber to 1 mM N-ethylmaleimide for 30s depressed isometric contractions in response to acetylcholine by 80%, while contractions induced by K+ and Ba2+ were reduced by less than 20% and 10%, respectively. Acetylcholine dose-response curves were shifted to the right while Ba2+ curves were unaffected. 4 It is suggested that N-ethylmaleimide has a selective effect on muscarinic responses in the longitudinal muscle by disrupting processes occurring after receptor occupancy but before the induction of phospholipid turnover or calcium influx in the postsynaptic membrane. PMID:7126999

4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes.

PubMed

Madsen, Ulf; Pickering, Darryl S; Nielsen, Birgitte; Bräuner-Osborne, Hans

2005-01-01

4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies, and the series of 4-alkyl-HIBO analogues have been extended in this paper in the search for versatile agents. Pharmacological characterization of five new analogues, branched and unbranched 4-alkyl-HIBO analogues, have been carried out. The present compounds are all weak antagonists at Group I mGluRs (mGluR1 and 5) presenting only small differences in potencies (Ki values ranging from 89 to 670 microM). Affinities were studied at native and cloned iGluRs, and the compounds described show preference for the AMPA receptor subtypes GluR1 and 2 over GluR3 and 4. However, compared to previous 4-alkyl-HIBO analogues, these compounds show a remarkably high affinity for the Kain preferring subtype GluR5. The observed GluR5 affinities were either similar or higher compared to their GluR1 and 2 affinity. Isopropyl-HIBO showed the highest affinity for GluR5 (Ki=0.16 microM), and represents a unique compound with high affinity towards the three subtypes GluR1, 2 and 5. In general, these compounds represent new selectivity profiles compared to previously reported Glu receptor analogues.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

PubMed Central

Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

2007-01-01

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

Diagnostic approach to hemoglobins with high oxygen affinity: experience from France and Belgium and review of the literature.

PubMed

Orvain, Corentin; Joly, Philippe; Pissard, Serge; Badiou, Stéphanie; Badens, Catherine; Bonello-Palot, Nathalie; Couque, Nathalie; Gulbis, Béatrice; Aguilar-Martinez, Patricia

2017-02-01

Congenital causes of erythrocytosis are now more easily identified due to the improvement of the molecular characterization of many of them. Among these causes, hemoglobins with high oxygen affinity take a large place. The aim of this work was to reevaluate the diagnostic approach of these disorders. To assess the current practices, we sent a questionnaire to the expert laboratories in the diagnosis of hemoglobinopathies in France and Belgium. In parallel, we gathered the methods used for the diagnosis of the hemoglobins with high oxygen affinity indexed in the international database HbVar. Even though they remain a rare cause of erythrocytosis (1 to 5 positive diagnosis every year in each of the questioned specialized laboratories), hemoglobins with high oxygen affinity are increasingly suspected by clinicians. Phenotypic assessment by laboratory techniques remains a main step in their diagnosis as it enables the finding of 93% of them in the questioned laboratories (28 of the 30 variants diagnosed during the last 5 years). Among the 96 hemoglobin variants with high oxygen affinity indexed in the international database, 87% could be diagnosed with phenotypic techniques. A direct measure of the p50 with the Hemox-Analyzer is included in the diagnostic approach of half of the laboratories only, because of the poor availability of this apparatus. Comparatively, the estimation of p50 by blood gas analyzers on venous blood is a much more convenient and attractive method but due to the lack of proof as to its effectiveness in the diagnosis of hemoglobins with high oxygen affinity, it requires further investigations. Beta- and alphaglobin genes analysis by molecular biology techniques is essential as it either allows a quick and definite identification of the variant or definitely excludes the diagnosis. It is thus systematically performed as a first or second step method, according to the laboratory practice.

Bimodal imprint chips for peptide screening: integration of high-throughput sequencing by MS and affinity analyses by surface plasmon resonance imaging.

PubMed

Wang, Weizhi; Li, Menglin; Wei, Zewen; Wang, Zihua; Bu, Xiangli; Lai, Wenjia; Yang, Shu; Gong, He; Zheng, Hui; Wang, Yuqiao; Liu, Ying; Li, Qin; Fang, Qiaojun; Hu, Zhiyuan

2014-04-15

Peptide probes and drugs have widespread applications in disease diagnostics and therapy. The demand for peptides ligands with high affinity and high specificity toward various targets has surged in the biomedical field in recent years. The traditional peptide screening procedure involves selection, sequencing, and characterization steps, and each step is manual and tedious. Herein, we developed a bimodal imprint microarray system to embrace the whole peptide screening process. Silver-sputtered silicon chip fabricated with microwell array can trap and pattern the candidate peptide beads in a one-well-one-bead manner. Peptides on beads were photocleaved in situ. A portion of the peptide in each well was transferred to a gold-coated chip to print the peptide array for high-throughput affinity analyses by surface plasmon resonance imaging (SPRi), and the peptide left in the silver-sputtered chip was ready for in situ single bead sequencing by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the bimodal imprint chip system, affinity peptides toward AHA were efficiently screened out from the 7 × 10(4) peptide library. The method provides a solution for high efficiency peptide screening.

Development of proteome-wide binding reagents for research and diagnostics.

PubMed

Taussig, Michael J; Schmidt, Ronny; Cook, Elizabeth A; Stoevesandt, Oda

2013-12-01

Alongside MS, antibodies and other specific protein-binding molecules have a special place in proteomics as affinity reagents in a toolbox of applications for determining protein location, quantitative distribution and function (affinity proteomics). The realisation that the range of research antibodies available, while apparently vast is nevertheless still very incomplete and frequently of uncertain quality, has stimulated projects with an objective of raising comprehensive, proteome-wide sets of protein binders. With progress in automation and throughput, a remarkable number of recent publications refer to the practical possibility of selecting binders to every protein encoded in the genome. Here we review the requirements of a pipeline of production of protein binders for the human proteome, including target prioritisation, antigen design, 'next generation' methods, databases and the approaches taken by ongoing projects in Europe and the USA. While the task of generating affinity reagents for all human proteins is complex and demanding, the benefits of well-characterised and quality-controlled pan-proteome binder resources for biomedical research, industry and life sciences in general would be enormous and justify the effort. Given the technical, personnel and financial resources needed to fulfil this aim, expansion of current efforts may best be addressed through large-scale international collaboration. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Second Generation Grp94-Selective Inhibitors Provide Opportunities for the Inhibition of Metastatic Cancer

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

Crowley, Vincent M.; Huard, Dustin J. E.; Lieberman, Raquel L.

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum (ER) resident isoform of the 90 kDa heat shock protein (Hsp90) family and its inhibition represents a promising therapeutic target for the treatment of many diseases. Modification of the first generation cis-amide bioisostere imidazole to alter the angle between the resorcinol ring and the benzyl side chain via cis-amide replacements produced compounds with improved Grp94 affinity and selectivity. Structure–activity relationship studies led to the discovery of compound 30, which exhibits 540 nm affinity and 73-fold selectivity towards Grp94. Grp94 is responsible for the maturation and trafficking of proteins associated with cellmore » signaling and motility, including select integrins. The Grp94-selective inhibitor 30 was shown to exhibit potent anti-migratory effects against multiple aggressive and metastatic cancers.« less