Expanding RNA binding specificity and affinity of engineered PUF domains.

PubMed

Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-05-18

Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

Expanding RNA binding specificity and affinity of engineered PUF domains

PubMed Central

Zhao, Yang-Yang; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-01-01

Abstract Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way. PMID:29490074

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.

Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.

PubMed

Kaplan, Jenifer B; Reinke, Aaron W; Keating, Amy E

2014-07-01

The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides. © 2014 The Protein Society.

DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

PubMed Central

Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

2009-01-01

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

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.

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

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

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.

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

Adherence of oral streptococci: evidence for nonspecific adsorption to saliva-coated hydroxylapatite surfaces.

PubMed Central

Staat, R H; Peyton, J C

1984-01-01

It is proposed that binding of oral streptococci to saliva-coated hydroxylapatite (SHA) surfaces is a multifactorial process involving both specific and nonspecific receptors. In this context, specific binding is described as a high-affinity, saturable interaction between the cell and binding surface. Conversely, nonspecific binding is considered to be a nonsaturable, generalized, low-affinity reaction. Experimental differentiation of specific binding from nonspecific binding was achieved with a competition assay which utilized a large excess of nonradiolabeled bacteria to compete with the 3H-labeled cells for attachment to receptors on 1.5 mg of SHA crystals. Competition assays of Streptococcus sanguis and Streptococcus mitis adhesion clearly demonstrated that the total binding isotherm was composed of a saturable specific binding reaction and a minor nonspecific binding component. This was further substantiated by analysis of nonlinear Scatchard plots of the total binding data. The competition data for Streptococcus mutans binding indicated that ca. 50% of the S. mutans binding appeared to be specific, although saturation of the SHA surfaces with bacterial cells could not be demonstrated. Experiments measuring desorption of radiolabeled cells from SHA crystals into buffer showed that ca. 50% of the bound S. mutans cells were removed after 4 h, whereas less than 5% of the S. sanguis cells were eluted from the SHA surfaces. The kinetics of attachment were studied by using an extract of Persea americana as a noncompetitive inhibitor of adherence. The total cell binding data for these experiments suggested a very rapid binding reaction followed by a slower rate of attachment. It was concluded from these three different experimental approaches that adherence of selected oral streptococci to SHA surfaces involves specific, high-affinity and nonspecific, low-affinity binding reactions. The concept is developed that in vitro streptococcal attachment to SHA can be described as a two-reaction process in which the low-affinity interaction of the cell with the SHA surface precedes the establishment of the stronger, specific bonds needed for the maintenance of streptococci in the oral cavity. PMID:6327530

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

Insulation and wiring specificity of BceR-like response regulators and their target promoters in Bacillus subtilis.

PubMed

Fang, Chong; Nagy-Staroń, Anna; Grafe, Martin; Heermann, Ralf; Jung, Kirsten; Gebhard, Susanne; Mascher, Thorsten

2017-04-01

BceRS and PsdRS are paralogous two-component systems in Bacillus subtilis controlling the response to antimicrobial peptides. In the presence of extracellular bacitracin and nisin, respectively, the two response regulators (RRs) bind their target promoters, P bceA or P psdA , resulting in a strong up-regulation of target gene expression and ultimately antibiotic resistance. Despite high sequence similarity between the RRs BceR and PsdR and their known binding sites, no cross-regulation has been observed between them. We therefore investigated the specificity determinants of P bceA and P psdA that ensure the insulation of these two paralogous pathways at the RR-promoter interface. In vivo and in vitro analyses demonstrate that the regulatory regions within these two promoters contain three important elements: in addition to the known (main) binding site, we identified a linker region and a secondary binding site that are crucial for functionality. Initial binding to the high-affinity, low-specificity main binding site is a prerequisite for the subsequent highly specific binding of a second RR dimer to the low-affinity secondary binding site. In addition to this hierarchical cooperative binding, discrimination requires a competition of the two RRs for their respective binding site mediated by only slight differences in binding affinities. © 2016 John Wiley & Sons Ltd.

beta. -Adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

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

van Koppen, C.J.; Hermanussen, M.W.; Verrijp, K.N.

1987-06-29

Specific binding of (/sup 125/I)-(-)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K/sub d/ = 5.3 +/- 0.9 pmol/l and R/sub T/ = 78 +/- 7 fmol/g tissue). The ..beta../sub 1/-selective antagonists atenolol and LK 203-030 inhibited specific (/sup 125/I)-(-)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous BETA/sub 2/-adrenoceptor population. In one subject using LK 203-030 a small ..beta../sub 1/-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK/sub H/-more » and pK/sub L/-values for the high and low affinity sites were 8.0 +/- 0.2 and 5.9 +/- 0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD/sub 2/-value of 6.63 +/- 0.19. 32 references, 4 figures, 2 tables.« less

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.

Mass-transport limitations in spot-based microarrays.

PubMed

Zhao, Ming; Wang, Xuefeng; Nolte, David

2010-09-20

Mass transport of analyte to surface-immobilized affinity reagents is the fundamental bottleneck for sensitive detection in solid-support microarrays and biosensors. Analyte depletion in the volume adjacent to the sensor causes deviation from ideal association, significantly slows down reaction kinetics, and causes inhomogeneous binding across the sensor surface. In this paper we use high-resolution molecular interferometric imaging (MI2), a label-free optical interferometry technique for direct detection of molecular films, to study the inhomogeneous distribution of intra-spot binding across 100 micron-diameter protein spots. By measuring intra-spot binding inhomogeneity, reaction kinetics can be determined accurately when combined with a numerical three-dimensional finite element model. To ensure homogeneous binding across a spot, a critical flow rate is identified in terms of the association rate k(a) and the spot diameter. The binding inhomogeneity across a spot can be used to distinguish high-affinity low-concentration specific reactions from low-affinity high-concentration non-specific binding of background proteins.

Complex high affinity interactions occur between MHCI and superantigens

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

1998-01-01

Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

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

Taketazu, F.; Chiba, S.; Shibuya, K.

1991-02-01

The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

PubMed Central

Honey, Denise M.; Best, Annie; Qiu, Huawei

2018-01-01

ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

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

Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

PubMed

Allevato, Michael; Bolotin, Eugene; Grossman, Mark; Mane-Padros, Daniel; Sladek, Frances M; Martinez, Ernest

2017-01-01

The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

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

PubMed

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

2000-06-15

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

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

PubMed

Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

2008-08-22

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

Treesearch

Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

2001-01-01

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

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.

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

Genome-wide inference of transcription factor-DNA binding specificity in cell regeneration using a combination strategy.

PubMed

Wang, Xiaofeng; Zhang, Aiqun; Ren, Weizheng; Chen, Caiyu; Dong, Jiahong

2012-11-01

The cell growth, development, and regeneration of tissue and organ are associated with a large number of gene regulation events, which are mediated in part by transcription factors (TFs) binding to cis-regulatory elements involved in the genome. Predicting the binding affinity and inferring the binding specificity of TF-DNA interactions at the genomic level would be fundamentally helpful for our understanding of the molecular mechanism and biological implication underlying sequence-specific TF-DNA recognition. In this study, we report the development of a combination method to characterize the interaction behavior of a 11-mer oligonucleotide segment and its mutations with the Gcn4p protein, a homodimeric, basic leucine zipper TF, and to predict the binding affinity and specificity of potential Gcn4p binders in the genome-wide scale. In this procedure, a position-mutated energy matrix is created based on molecular modeling analysis of native and mutated Gcn4p-DNA complex structures to describe the position-independent interaction energy profile of Gcn4p with different nucleotide types at each position of the oligonucleotide, and the energy terms extracted from the matrix and their interactives are then correlated with experimentally measured affinities of 19268 distinct oligonucleotides using statistical modeling methodology. Subsequently, the best one of built regression models is successfully applied to screen those of potential high-affinity Gcn4p binders from the complete genome. The findings arising from this study are briefly listed below: (i) The 11 positions of oligonucleotides are highly interactive and non-additive in contribution to Gcn4p-DNA binding affinity; (ii) Indirect conformational effects upon nucleotide mutations as well as associated subtle changes in interfacial atomic contacts, but not the direct nonbonded interactions, are primarily responsible for the sequence-specific recognition; (iii) The intrinsic synergistic effects among the sequence positions of oligonucleotides determine Gcn4p-DNA binding affinity and specificity; (iv) Linear regression models in conjunction with variable selection seem to perform fairly well in capturing the internal dependences hidden in the Gcn4p-DNA system, albeit ignoring nonlinear factors may lead the models to systematically underestimate and overestimate high- and low-affinity samples, respectively. © 2012 John Wiley & Sons A/S.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

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

Wang, Deng-Liang; Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou; Song, Yan-Ling

2014-10-31

Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the idealmore » antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.« less

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

Two classes of receptor specific for sperm-activating peptide III in sand-dollar spermatozoa.

PubMed

Yoshino, K; Suzuki, N

1992-06-15

We characterized receptors specific for sperm-activating peptide III (SAP-III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross-linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP-III analogueto C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP-III in the spermatozoa. One of the receptors (high-affinity) had a Kd of 3.4 nM and 3.4 x 10(4) binding sites/spermatozoon. The other receptor (low-affinity) had a Kd of 48 nM, with 6.1 x 10(4) binding sites/spermatozoon. The Kd of the high-affinity receptor was comparable to the median effective concentration of the intracellular-pH-increasing activity of SAP-III and that of the low-affinity receptor was comparable to the median effective concentration of the cellular-cGMP-elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high-affinity members. Similar results were also obtained from a binding experiment using a sperm-membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP-III analogue and a chemical cross-linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.

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.

Batrachotoxin Changes the Properties of the Muscarinic Receptor in Rat Brain and Heart: Possible Interaction(s) between Muscarinic Receptors and Sodium Channels

NASA Astrophysics Data System (ADS)

Cohen-Armon, Malca; Kloog, Yoel; Henis, Yoav I.; Sokolovsky, Mordechai

1985-05-01

The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).

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.

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

Use of polyclonal and monoclonal antibodies to study hCG-receptor interactions

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

Milius, R.P.

1985-01-01

Although the glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) bind to different receptors, each contains an identical alpha subunit. Specificity is somehow endowed by theta subunits which are distinct for each hormone. Human choriogonadotropin (hCG) is a natural LH analog that contains a beta subunit nearly identical to that of LH. The roles of these subunits in the recognition and high affinity binding of hCG to receptor was examined. Polyclonal and monoclonal antibodies specific for the individual subunits of hCG were used to probe the hormone-receptor interaction. Conformation-specific and sequence-specific antibodies were examined for their abilities to bindmore » Triton X-100-solubilized /sup 125/I-hCG-receptor complex and to inhibit hormone binding to crude rat ovarian membranes containing receptor. Even though the immunoreactive sites are not located on the receptor binding surface of the beta subunit, most, but not all, of these polyclonal and monoclonal antibodies were able to inhibit /sup 125/I-hCG binding to receptor. Although the inhibition of binding may be due to steric interference due to the size of the antibody molecules, a two-step model for hCG binding to receptor is presented that also explains these results. In this model, the beta subunit initially binds with the receptor with a highly specific but low affinity interaction. This activates a site for the high affinity binding of the alpha subunit and stabilization of the complex. This is an attractive model as it may be applied to other glycoprotein hormones sharing an alpha subunit.« less

Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

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

D'Amato, R.J.; Largent, B.L.; Snowman, A.M.

1987-07-01

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine bindingmore » reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.« less

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

PubMed Central

Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

2003-01-01

A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

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

PubMed Central

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

1991-01-01

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

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

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

NASA Astrophysics Data System (ADS)

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-03-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

PubMed Central

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-01-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071–40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands. PMID:24670678

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

Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode

PubMed Central

Cole, David K.; Sami, Malkit; Scott, Daniel R.; Rizkallah, Pierre J.; Borbulevych, Oleg Y.; Todorov, Penio T.; Moysey, Ruth K.; Jakobsen, Bent K.; Boulter, Jonathan M.; Baker, Brian M.; Yi Li

2013-01-01

Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A∗0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3β loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies. PMID:23805144

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

Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

PubMed

Neupane, Durga P; Avalos, Dante; Fullam, Stephanie; Roychowdhury, Hridindu; Yukl, Erik T

2017-10-20

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

PubMed

Thekkumkara, Thomas; Snyder, Russell; Karamyan, Vardan T

2016-01-01

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

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

PubMed

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

2012-01-01

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

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

PubMed Central

Jackson, Abby J.; Anguizola, Jeanethe; Pfaunmiller, Erika L.; Hage, David S.

2013-01-01

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions. PMID:23657448

Proteome-wide inference of human endophilin 1-binding peptides.

PubMed

Wu, Gang; Zhang, Zeng-Li; Fu, Chun-Jiang; Lv, Feng-Lin; Tian, Fei-Fei

2012-10-01

Human endophilin 1 (hEndo1) is a multifunctional protein that was found to bind a wide spectrum of prolinerich endocytic proteins through its Src homology 3 (SH3) domain. In order to elucidate the unknown biological functions of hEndo1, it is essential to find out the cytoplasmic components that hEndo1 recognizes and binds. However, it is too time-consuming and expensive to synthesize all peptide candidates found in the human proteome and to perform hEndo1 SH3-peptide affinity assay to identify the hEndo1-binding partners. In the present work, we describe a structure/ sequence-hybrid approach to perform proteome-wide inference of human hEndo1-binding peptides using the information gained from both the primary sequence of affinity-known peptides and the interaction profile involved in hEndo1 SH3-peptide complex three-dimensional structures. Modeling results show that (i) different residue positions contribute distinctly to peptide affinity and specificity; P-1, P2 and P4 are most important, P1 and P3 are also effective, and P-3, P-2, P0, P5 and P6 are relatively insignificant, (ii) the consensus core PXXP motif is necessary but not sufficient for determining high affinity of peptides, and some other positions must be also essential in the hEndo1 SH3-peptide binding, and (iii) the alternating arrangement of polar and nonpolar amino acids along peptide sequence is critical for the high specificity of peptide recognition by hEndo1 SH3 domain. In addition, we also find that the residue type at a specific position of hEndo1-binding peptides is not stringently invariable; amino acids that possess similar polarity could replace each other without substantial influence on peptide affinity. In this way, hEndo1 presents a broad specificity in the peptide ligands that it binds.

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.

Identification of lanthanum-specific peptides for future recycling of rare earth elements from compact fluorescent lamps.

PubMed

Lederer, Franziska L; Curtis, Susan B; Bachmann, Stefanie; Dunbar, W Scott; MacGillivray, Ross T A

2017-05-01

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost-efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO 4 :Ce 3+ ,Tb 3+ (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 10 9 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptide's affinity for the fluorescent phosphor components CAT (CeMgAl 11 O 19 :Tb 3+ ) and BAM (BaMgAl 10 O 17 :Eu 2+ ). No affinity was found for other fluorescent phosphor components such as YOX (Y 2 O 3 :Eu 3+ ). The binding specificity of the RCQYPLCS peptide loop was improved 3-51-fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016-1024. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1.

PubMed

DeJong, Eric S; Chang, Chia-en; Gilson, Michael K; Marino, John P

2003-07-08

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.

Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers

PubMed Central

Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

2015-01-01

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria. PMID:25884791

Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers.

PubMed

Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

2015-04-15

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.

An Evolutionary/Biochemical Connection Between Promoter- and Primer-Dependent Polymerases Revealed by Selective Evolution of Ligands by Exponential Enrichment (SELEX).

PubMed

Fenstermacher, Katherine J; Achuthan, Vasudevan; Schneider, Thomas D; DeStefano, Jeffrey J

2018-01-16

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified Selective Evolution of Ligands by Exponential Enrichment (SELEX) approach. Two Taq -specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. Taq1 contained 8 nucleotides (5' -CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCR reactions. Similarly, exonuclease minus Klenow polymerase also selected a high affinity primer that contained a related core promoter sequence from phage T7 RNAP (5' -ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity suggesting that binding was highly sequence-specific. The results are discussed in the context of possible effects on multi-primer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs. Importance This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function, or be a consequences of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promotors. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology. Copyright © 2018 American Society for Microbiology.

A Novel High-Mannose Specific Lectin from the Green Alga Halimeda renschii Exhibits a Potent Anti-Influenza Virus Activity through High-Affinity Binding to the Viral Hemagglutinin

PubMed Central

Mu, Jinmin; Hirayama, Makoto; Sato, Yuichiro; Morimoto, Kinjiro; Hori, Kanji

2017-01-01

We have isolated a novel lectin, named HRL40 from the green alga Halimeda renschii. In hemagglutination-inhibition test and oligosaccharide-binding experiment with 29 pyridylaminated oligosaccharides, HRL40 exhibited a strict binding specificity for high-mannose N-glycans having an exposed (α1-3) mannose residue in the D2 arm of branched mannosides, and did not have an affinity for monosaccharides and other oligosaccharides examined, including complex N-glycans, an N-glycan core pentasaccharide, and oligosaccharides from glycolipids. The carbohydrate binding profile of HRL40 resembled those of Type I high-mannose specific antiviral algal lectins, or the Oscillatoria agardhii agglutinin (OAA) family, which were previously isolated from red algae and a blue-green alga (cyanobacterium). HRL40 potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells with half-maximal effective dose (ED50) of 2.45 nM through high-affinity binding to a viral envelope hemagglutinin (KD, 3.69 × 10−11 M). HRL40 consisted of two isolectins (HRL40-1 and HRL40-2), which could be separated by reverse-phase HPLC. Both isolectins had the same molecular weight of 46,564 Da and were a disulfide -linked tetrameric protein of a 11,641 Da polypeptide containing at least 13 half-cystines. Thus, HRL40, which is the first Type I high-mannose specific antiviral lectin from the green alga, had the same carbohydrate binding specificity as the OAA family, but a molecular structure distinct from the family. PMID:28813016

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics.

PubMed

Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison E; Han, Jongyoon; Alter, Galit

2016-03-30

Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary 'bind-elute' separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets-cells or proteins-bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients.

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

PubMed Central

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

2013-01-01

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

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

Crystal structure of a cocaine-binding antibody.

PubMed

Larsen, N A; Zhou, B; Heine, A; Wirsching, P; Janda, K D; Wilson, I A

2001-08-03

Murine monoclonal antibody GNC92H2 was elicited by active immunization with a cocaine immunoconjugate and binds free cocaine with excellent specificity and moderate affinity. Improvement of affinity, as well as humanization of GNC92H2, would be advantageous in immunopharmacotherapy for cocaine addiction, and for emergency cases of drug overdose. Toward this end, the crystal structure of an engineered murine-human chimeric Fab of GNC92H2 complexed with cocaine was determined at 2.3 A resolution. Structural analysis reveals a binding pocket with high shape and charge complementarity to the cocaine framework, which explains the specificity for cocaine, as opposed to the pharmacologically inactive cocaine metabolites. Importantly, the structure provides a foundation for mutagenesis to enhance the binding affinity for cocaine and potent cocaine derivatives, such as cocaethylene, and for additional humanization of the antibody. Copyright 2001 Academic Press.

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

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.

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

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

PubMed

Epps, D E; Raub, T J; Caiolfa, V; Chiari, A; Zamai, M

1999-01-01

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

[High non-specific binding of the beta(1) -selective radioligand 2-(125)I-ICI-H].

PubMed

Riemann, B; Law, M P; Kopka, K; Wagner, St; Luthra, S; Pike, V W; Neumann, J; Kirchhefer, U; Schmitz, W; Schober, O; Schäfers, M

2003-08-01

As results of cardiac biopsies suggest, myocardial beta(1) -adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac beta(2)-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess beta-adrenoceptors non-invasively. Among the novel racemic analogues of the established beta(1)-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to beta(1)-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative sub-type selective beta(1)-adrenergic radioligand in cardiac imaging. Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac beta-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-(125)I-ICI-H from the silylated precursor 2-SiMe(3)-ICI-H was established. The specific activity was 80 GBq/ micro mol, the radiochemical yield ranged from 70 to 80%. The unlabelled compound 2-I-ICI-H showed high beta(1)-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac beta-adrenoceptors. The radiolabelled counterpart 2-(125)I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac beta(1)-adrenoceptors in vivo. Because of its high non-specific binding 2-(125)I-ICI-H is no suitable radiotracer for imaging in vivo.

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

Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

PubMed Central

Williams, Chad M.; Schonnesen, Alexandra A.; Zhang, Shu-Qi; Ma, Ke-Yue; He, Chenfeng; Yamamoto, Tori; Eckhardt, S. Gail; Klebanoff, Christopher A.; Jiang, Ning

2017-01-01

The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously established TCR discovery platform using 2D TCR affinity and sequence test would allow for selection of TCRs specific to any given antigen with the desirable attributes of high TCR affinity, CD8 co-receptor independence and functional superiority. Utilizing TCRs with less CD8 contribution could be beneficial for adoptive cell transfer immunotherapies using naturally occurring or genetically engineered T cells against viral or cancer-associated antigens. PMID:28804489

Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung.

PubMed Central

Walsh, D A; Salmon, M; Featherstone, R; Wharton, J; Church, M K; Polak, J M

1994-01-01

1. The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]-Bolton Hunter-labelled substance P ([125I]-BH-SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung. 2. [125I]-BH-SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung. 3. Specific binding of [125I]-BH-SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150-800 microns and was less dense in trachea than in main bronchi. 4. [125I]-BH-SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125I]-BH-SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors. 5. In both human and guinea pig lung, [125I]-BH-SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]-BH-SP binding sites in both guinea pig arteries and bronchi.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:7534186

High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

PubMed Central

Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

2015-01-01

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

PubMed

Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

2015-02-13

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

High affinity ligands from in vitro selection: Complex targets

PubMed Central

Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

1998-01-01

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

PubMed

Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

2006-12-21

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

PubMed Central

Jenkins, Jeremy L; Dean, Donald H

2001-01-01

Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

USGS Publications Warehouse

Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

2000-01-01

High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

Two distinctive β subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensis.

PubMed

Bao, Haibo; Liu, Yang; Zhang, Yixi; Liu, Zewen

2017-08-01

Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one β subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third β subunits (Locβ3) was identified in this study, which reveals at least three β subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with K d values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locβ1, Locβ2 and Locβ3 respectively. Specific immunodepletion of Locβ1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locβ3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locβ2 had no significant effect on the specific [ 3 H]imidacloprid binding. Taken together, these data indicated that Locβ1 and Locβ3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlβ1 was in two binding sites for imidacloprid. The involvement of two β subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in β subunits in L. migratoria. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure–function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8

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

Matho, Michael H.; Schlossman, Andrew; Gilchuk, Iuliia M.

Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here in this paper, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, andmore » VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138– and VACV-304–binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.« less

Structure–function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8

DOE PAGES

Matho, Michael H.; Schlossman, Andrew; Gilchuk, Iuliia M.; ...

2017-11-09

Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here in this paper, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, andmore » VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138– and VACV-304–binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.« less

[125I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

PubMed Central

Dumont, Yvan; Quirion, Rémi

2000-01-01

GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method. [125I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [125I]-GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [125I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA. Autoradiographic studies demonstrated that [125I]-GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [125I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y4 receptor subtype. In summary, [125I]-GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]-GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype. PMID:10694200

Channel architecture in maltoporin: dominance studies with lamB mutations influencing maltodextrin binding provide evidence for independent selectivity filters in each subunit.

PubMed Central

Ferenci, T; Lee, K S

1989-01-01

Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits. PMID:2521623

Integration of element specific persistent homology and machine learning for protein-ligand binding affinity prediction.

PubMed

Cang, Zixuan; Wei, Guo-Wei

2018-02-01

Protein-ligand binding is a fundamental biological process that is paramount to many other biological processes, such as signal transduction, metabolic pathways, enzyme construction, cell secretion, and gene expression. Accurate prediction of protein-ligand binding affinities is vital to rational drug design and the understanding of protein-ligand binding and binding induced function. Existing binding affinity prediction methods are inundated with geometric detail and involve excessively high dimensions, which undermines their predictive power for massive binding data. Topology provides the ultimate level of abstraction and thus incurs too much reduction in geometric information. Persistent homology embeds geometric information into topological invariants and bridges the gap between complex geometry and abstract topology. However, it oversimplifies biological information. This work introduces element specific persistent homology (ESPH) or multicomponent persistent homology to retain crucial biological information during topological simplification. The combination of ESPH and machine learning gives rise to a powerful paradigm for macromolecular analysis. Tests on 2 large data sets indicate that the proposed topology-based machine-learning paradigm outperforms other existing methods in protein-ligand binding affinity predictions. ESPH reveals protein-ligand binding mechanism that can not be attained from other conventional techniques. The present approach reveals that protein-ligand hydrophobic interactions are extended to 40Å  away from the binding site, which has a significant ramification to drug and protein design. Copyright © 2017 John Wiley & Sons, Ltd.

High-resolution mapping of transcription factor binding sites on native chromatin

PubMed Central

Kasinathan, Sivakanthan; Orsi, Guillermo A.; Zentner, Gabriel E.; Ahmad, Kami; Henikoff, Steven

2014-01-01

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions. PMID:24336359

Inherent limitations of probabilistic models for protein-DNA binding specificity

PubMed Central

Ruan, Shuxiang

2017-01-01

The specificities of transcription factors are most commonly represented with probabilistic models. These models provide a probability for each base occurring at each position within the binding site and the positions are assumed to contribute independently. The model is simple and intuitive and is the basis for many motif discovery algorithms. However, the model also has inherent limitations that prevent it from accurately representing true binding probabilities, especially for the highest affinity sites under conditions of high protein concentration. The limitations are not due to the assumption of independence between positions but rather are caused by the non-linear relationship between binding affinity and binding probability and the fact that independent normalization at each position skews the site probabilities. Generally probabilistic models are reasonably good approximations, but new high-throughput methods allow for biophysical models with increased accuracy that should be used whenever possible. PMID:28686588

Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

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

Besemer, J.; Hujber, A.; Kuhn, B.

1989-10-15

The interaction of {sup 125}I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4{degree}C to pH 3 resistance at 37{degree}C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37{degree}C led to nonlinearmore » curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.« less

Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses

PubMed Central

Grünewald, Nora; Salvatico, Charlotte; Kress, Vanessa

2018-01-01

Abstract The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells. PMID:29464196

Probing the human estrogen receptor-α binding requirements for phenolic mono- and di-hydroxyl compounds: A combined synthesis, binding and docking study

PubMed Central

McCullough, Christopher; Neumann, Terrence S.; Gone, Jayapal Reddy; He, Zhengjie; Herrild, Christian; Wondergem, Julie; Pandey, Rajesh K.; Donaldson, William A.; Sem, Daniel S.

2014-01-01

Various estrogen analogs were synthesized and tested for binding to human ERα using a fluorescence polarization displacement assay. Binding affinity and orientation were also predicted using docking calculations. Docking was able to accurately predict relative binding affinity and orientation for estradiol, but only if a tightly bound water molecule bridging Arg394/Glu353 is present. Di-hydroxyl compounds sometimes bind in two orientations, which are flipped in terms of relative positioning of their hydroxyl groups. Di-hydroxyl compounds were predicted to bind with their aliphatic hydroxyl group interacting with His524 in ERα. One nonsteroid-based dihdroxyl compound was 1000-fold specific for ERβ over ERα, and was also 25-fold specific for agonist ERβ versus antagonist activity. Docking predictions suggest this specificity may be due to interaction of the aliphatic hydroxyl with His475 in the agonist form of ERβ, versus with Thr299 in the antagonist form. But, the presence of this aliphatic hydroxyl is not required in all compounds, since mono-hydroxyl (phenolic) compounds bind ERα with high affinity, via hydroxyl hydrogen bonding interactions with the ERα Arg394/Glu353/water triad, and van der Waals interactions with the rest of the molecule. PMID:24315190

Binding of perlecan to transthyretin in vitro.

PubMed Central

Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

1997-01-01

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

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

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

PubMed

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

2002-07-01

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

High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses.

PubMed

Nürnberger, T; Nennstiel, D; Jabs, T; Sacks, W R; Hahlbrock, K; Scheel, D

1994-08-12

An oligopeptide of 13 amino acids (Pep-13) identified within a 42 kDa glycoprotein elicitor from P. mega-sperma was shown to be necessary and sufficient to stimulate a complex defense response in parsley cells comprising H+/Ca2+ influxes, K+/Cl- effluxes, an oxidative burst, defense-related gene activation, and phytoalexin formation. Binding of radiolabeled Pep-13 to parsley microsomes and protoplasts was specific, reversible, and saturable. Identical structural features of Pep-13 were found to be responsible for specific binding and initiation of all plant responses analyzed. The high affinity binding site recognizing the peptide ligand (KD = 2.4 nM) may therefore represent a novel class of receptors in plants, and the rapidly induced ion fluxes may constitute elements of the signal transduction cascade triggering pathogen defense in plants.

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

Zolpidem displays heterogeneity in its binding to the nonhuman primate benzodiazepine receptor in vivo.

PubMed

Schmid, L; Bottlaender, M; Fuseau, C; Fournier, D; Brouillet, E; Mazière, M

1995-10-01

The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [11C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [11C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and approximately 100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for < 32% of the specific [11C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.

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.

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

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

Lach, E.; Trifilieff, A.; Landry, Y.

1991-01-01

The binding of the radiolabeled bombesin analogue ({sup 125}I-Tyr{sup 4})bombesin to guinea-pig lung membranes was investigated. Binding of ({sup 125}I-Tyr{sup 4})bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B{sub max} = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K{sub D} = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as ({sup 125}I-Tyr{sup 4})bombesin, neuromedin B and neuromedin C inhibited the binding of ({sup 125}I-Tyr{sup 4})bombesin inmore » an order of potencies as follows: ({sup 125}I-Tyr{sup 4})bombesin {gt} bombesin {ge} neuromedin C {much gt} neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.« less

Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.

PubMed

Aidoudi, Sallouha; Bikfalvi, Andreas

2010-11-01

Platelet factor-4 (PF4), a platelet-derived chemokine, has two important functions in the vasculature. It has a pro-atherogenic role while also having anti-angiogenic effects. The activity of platelet factor-4 (PF4), unlike other chemokines that bind to specific receptors, depends on its unusually high affinity for proteoglycans and other negatively charged molecules. High affinity for heparan sulfates was thought to be central to all of PF4's biological functions. However, other mechanisms have been described such as direct growth factor binding, activation of the CXCR3B chemokine receptor isoform that is present in some vascular cells or binding to lipoprotein-related protein-1 (LRP1). Furthermore, PF4 also binds to integrins with affinities similar to matrix molecules. These interactions may explain the effects of PF4 in healthy and pathological tissues. However, the mechanisms involved in PF4's activity are complex and may depend on a given tissue or localisation. Overall, while much is already known about PF4, its specific role in atherosclerosis and angiogenesis remains still to be clarified.

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides

PubMed Central

Yoga, Yano M. K.; Traore, Daouda A. K.; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R.; Barker, Andrew; Leedman, Peter J.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

2012-01-01

Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5′-CCCTCCCT-3′ DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5′-ACCCCA-3′ DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad. PMID:22344691

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

PubMed

Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

2012-06-01

Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

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.

The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

USGS Publications Warehouse

Persson, Petra; Shrimpton, J. Mark; McCormick, Stephen D.; Bjornsson, Bjorn Thrandur

2000-01-01

High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol × mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol × mg protein−1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

High Affinity Binding of Epibatidine to Serotonin Type 3 Receptors*

PubMed Central

Drisdel, Renaldo C.; Sharp, Douglas; Henderson, Tricia; Hales, Tim G.; Green, William N.

2008-01-01

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of 125I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT3Rs) block the remaining 25%. 125I-Epibatidine binds with a high affinity to native 5-HT3Rs of N1E-115 cells and to receptors composed of only 5-HT3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT3R-specific antagonist MDL72222, and the 5-HT3R agonist chlorophenylbiguanide readily competed with 125I-epibatidine binding to 5-HT3Rs. Nicotine was a poor competitor for 125I-epibatidine binding to 5-HT3Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of 125I-epibatidine binding to 5-HT3Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT3Rs in neuroblastoma cell lines and 5-HT3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT3Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT3R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT3Rs. PMID:17702741

Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis.

PubMed

Paula, Stefan; Tabet, Michael R; Keenan, Susan M; Welsh, William J; Ball, W James

2003-01-17

Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabolites by [3H]-radioligand binding assays. mAb 3P1A6 (K(d) = 0.22 nM) displayed a 50-fold higher affinity for cocaine than mAb MM0240PA (K(d) = 11 nM) and also had a greater specificity for cocaine. For the systematic exploration of both antibodies' binding specificities, we used a set of approximately 35 cocaine analogues as structural probes by determining their relative binding affinities (RBAs) using an enzyme-linked immunosorbent competition assay. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models on the basis of comparative molecular field analysis (CoMFA) techniques correlated the binding data with structural features of the ligands. The analysis indicated that despite the mAbs' differing specificities for cocaine, the relative contributions of the steric (approximately 80%) and electrostatic (approximately 20%) field interactions to ligand-binding were similar. Generated three-dimensional CoMFA contour plots then located the specific regions about cocaine where the ligand/receptor interactions occurred. While the overall binding patterns of the two mAbs had many features in common, distinct differences were observed about the phenyl ring and the methylester group of cocaine. Furthermore, using previously published data, a 3D-QSAR model was developed for cocaine binding to the dopamine reuptake transporter (DAT) that was compared to the mAb models. Although the relative steric and electrostatic field contributions were similar to those of the mAbs, the DAT cocaine-binding site showed a preference for negatively charged ligands. Besides establishing molecular level insight into the interactions that govern cocaine binding specificity by biopolymers, the three-dimensional images obtained reflect the properties of the mAbs binding pockets and provide the initial information needed for the possible design of novel antibodies with properties optimized for immunotherapy. Copyright 2003 Elsevier Science Ltd.

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

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.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

PubMed

Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

2014-10-31

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2016-01-01

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

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

PubMed Central

Zhao, Wenjing; McCallum, Scott A.; Xiao, Zhongping; Zhang, Fuming; Linhardt, Robert J.

2011-01-01

Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs. PMID:21658003

Recent Progress in Aptamer-Based Functional Probes for Bioanalysis and Biomedicine.

PubMed

Zhang, Huimin; Zhou, Leiji; Zhu, Zhi; Yang, Chaoyong

2016-07-11

Nucleic acid aptamers are short synthetic DNA or RNA sequences that can bind to a wide range of targets with high affinity and specificity. In recent years, aptamers have attracted increasing research interest due to their unique features of high binding affinity and specificity, small size, excellent chemical stability, easy chemical synthesis, facile modification, and minimal immunogenicity. These properties make aptamers ideal recognition ligands for bioanalysis, disease diagnosis, and cancer therapy. This review highlights the recent progress in aptamer selection and the latest applications of aptamer-based functional probes in the fields of bioanalysis and biomedicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands

PubMed Central

2015-01-01

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors. PMID:25073654

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.

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.

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

PubMed

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

2005-10-15

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

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

PubMed

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

2016-10-01

Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. Copyright © 2016 Elsevier B.V. All rights reserved.

Use of 2-(/sup 125/I)iodomelatonin to characterize melatonin binding sites in chicken retina

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

Dubocovich, M.L.; Takahashi, J.S.

2-(/sup 125/I)Iodomelatonin binds with high affinity to a site possessing the pharmacological characteristics of a melatonin receptor in chicken retinal membranes. The specific binding of 2-(/sup 125/I)iodomelatonin is stable, saturable, and reversible. Saturation experiments indicated that 2-(/sup 125/I)iodomelatonin labeled a single class of sites with an affinity constant (Kd) of 434 +/- 56 pM and a total number of binding sites (Bmax) of 74.0 +/- 13.6 fmol/mg of protein. The affinity constant obtained from kinetic analysis was in close agreement with that obtained in saturation experiments. Competition experiments showed a monophasic reduction of 2-(/sup 125/I)iodomelatonin binding with a pharmacological ordermore » of indole amine affinities characteristic of a melatonin receptor: 2-iodomelatonin greater than 6-chloromelatonin greater than or equal to melatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 6-hydroxymelatonin greater than or equal to 6-methoxymelatonin much greater than N-acetyltryptamine greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine greater than 5-hydroxytryptamine (inactive). The affinities of these melatonin analogs in competing for 2-(/sup 125/I)iodomelatonin binding sites were correlated closely with their potencies for inhibition of the calcium-dependent release of (3H)dopamine from chicken and rabbit retinas, indicating association of the binding site with a functional response regulated by melatonin. The results indicate that 2-(/sup 125/I)iodomelatonin is a selective, high-affinity radioligand for the identification and characterization of melatonin receptor sites.« less

DNA-aptamers binding aminoglycoside antibiotics.

PubMed

Nikolaus, Nadia; Strehlitz, Beate

2014-02-21

Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

Development of a high specific activity radioligand, /sup 125/I-LSD, and its application to the study of serotonin receptors

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

Kadan, M.J.

/sup 125/I-Labeled receptor ligands can be synthesized with specific activities exceeding 2000 Ci/mmol, making them nearly 70-fold more sensitive in receptor site assays than (mono) tritiated ligands. We have synthesized and characterized /sup 125/I-lysergic acid diethylamide (/sup 125/I-LSD), the first radioiodinated ligand for serotonin receptor studies. The introduction of /sup 125/I at the 2 position of LSD increased both the affinity and selectivity of this compound for serotonin 5-HT/sub 2/ receptors in rat cortex. The high specific activity of /sup 125/I-LSD and its high ratio of specific to nonspecific binding make this ligand especially useful for autoradiographic studies of serotoninmore » receptor distribution. We have found that /sup 125/I-LSD binds with high affinity to a class of serotonin receptors in the CNS of the marine mollusk Aplysia californica.« less

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

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

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

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

Sine, Steven M.; Huang, Sun; Li, Shu-Xing

2013-09-01

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

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

Howard, A.D.

The aim of this research was to purify and characterize active opioid receptors and elucidate molecular aspects of opioid receptor heterogeneity. Purification to apparent homogeneity of an opioid binding protein from bovine caudate was achieved by solubilization in the non-ionic detergent, digitonin, followed by sequential chromatography on the opiate affinity matrix, ..beta..-naltrexylethylenediamine-CH-Sepharose 4B, and on the lectine affinity matrix, wheat germ agglutinin-agarose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) followed by autoradiography revealed that radioiodinated purified receptor gave a single band. Purified receptor preparations showed a specific activity of 12,000-15,000 fmol of opiate bound per mgmore » of protein. Radioiodinated human beta-endorphin (/sup 125/I-beta-end/sub H/) was used as a probe to investigate the ligand binding subunits of mu and delta opioid receptors. /sup 125/I-beta-end/sub H/ was shown to bind to a variety of opioid receptor-containing tissues with high affinity and specificity with preference for mu and delta sites, and with little, if any, binding to kappa sites. Affinity crosslinking techniques were employed to covalently link /sup 125/I-beta-end/sub H/ to opioid receptors, utilizing derivatives of bis-succinimidyl esters that are bifunctional crosslinkers with specificities for amino and sulfhydryl groups. This, and competition experiments with high type-selective ligands, permitted the assignment of two labeled peptides to their receptor types, namely a peptide of M/sub r/ = 65,000 for mu receptors and one of M/sub r/ = 53,000 for delta receptors.« less

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

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

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

1991-05-01

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

Extreme disorder in an ultrahigh-affinity protein complex

NASA Astrophysics Data System (ADS)

Borgia, Alessandro; Borgia, Madeleine B.; Bugge, Katrine; Kissling, Vera M.; Heidarsson, Pétur O.; Fernandes, Catarina B.; Sottini, Andrea; Soranno, Andrea; Buholzer, Karin J.; Nettels, Daniel; Kragelund, Birthe B.; Best, Robert B.; Schuler, Benjamin

2018-03-01

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

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

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinitymore » to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.« less

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

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.

Ecdysteroid receptors in Drosophila melanogaster adult females

USDA-ARS?s Scientific Manuscript database

Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melanogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent w...

Customizing Monoclonal Antibodies for the Treatment of Methamphetamine Abuse: Current and Future Applications

PubMed Central

Peterson, Eric C.; Gentry, W. Brooks

2015-01-01

Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse, and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggest a scientific vision for how intact mAb (singular and plural) or small antigen binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution x-ray crystal structure of a high affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen binding fragments (Fab) and single chain variable fragments (scFv) are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which the METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites and neuronal connections. PMID:24484976

Customizing monoclonal antibodies for the treatment of methamphetamine abuse: current and future applications.

PubMed

Peterson, Eric C; Gentry, W Brooks; Owens, S Michael

2014-01-01

Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggests a scientific vision for how intact monoclonal antibody (mAb) (singular and plural) or small antigen-binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review, we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution X-ray crystal structure of a high-affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen-binding fragments and single-chain variable fragments are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites, and neuronal connections. © 2014 Elsevier Inc. All rights reserved.

In vivo evaluation of radiotracers targeting the melanin-concentrating hormone receptor 1: [11C]SNAP-7941 and [18F]FE@SNAP reveal specific uptake in the ventricular system.

PubMed

Zeilinger, Markus; Dumanic, Monika; Pichler, Florian; Budinsky, Lubos; Wadsak, Wolfgang; Pallitsch, Katharina; Spreitzer, Helmut; Lanzenberger, Rupert; Hacker, Marcus; Mitterhauser, Markus; Philippe, Cécile

2017-08-14

The MCHR1 is involved in the regulation of energy homeostasis and changes of the expression are linked to a variety of associated diseases, such as diabetes and adiposity. The study aimed at the in vitro and in vivo evaluation of [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP as potential PET-tracers for the MCHR1. Competitive binding studies with non-radioactive derivatives and small-animal PET/CT and MRI brain studies were performed under baseline conditions and tracer displacement with the unlabelled MCHR1 antagonist (±)-SNAP-7941. Binding studies evinced high binding affinity of the non-radioactive derivatives. Small-animal imaging of [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP evinced high tracer uptake in MCHR1-rich regions of the ventricular system. Quantitative analysis depicted a significant tracer reduction after displacement with (±)-SNAP-7941. Due to the high binding affinity of the non-labelled derivatives and the high specific tracer uptake of [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP, there is strong evidence that both radiotracers may serve as highly suitable agents for specific MCHR1 imaging.

Adrenergic receptors in frontal cortex in human brain.

PubMed

Cash, R; Raisman, R; Ruberg, M; Agid, Y

1985-02-05

The binding of three adrenergic ligands ([3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol) was studied in the frontal cortex of human brain. alpha 1-Receptors, labeled by [3H]prazosin, predominated. [3H]Clonidine bound to two classes of sites, one of high affinity and one of low affinity. Guanosine triphosphate appeared to lower the affinity of [3H]clonidine for its receptor. [3H]Dihydroalprenolol bound to three classes of sites: the beta 1-receptor, the beta 2-receptor and a receptor with low affinity which represented about 40% of the total binding, but which was probably a non-specific site; the beta 1/beta 2 ratio was 1/2.

An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis

DOE PAGES

Kariolis, Mihalis S.; Miao, Yu Rebecca; Jones, Douglas S.; ...

2014-09-21

Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl ‘decoy receptor’ that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high affinity Axl variant caused structural alterations in side chains across the Gas6/Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc-fusion, the engineered decoy receptor bound tomore » Gas6 with femtomolar affinity, an 80-fold improvement compared to the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Additionally, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.« less

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

PubMed Central

Hay, Iain D.; Du, Jinping; Burr, Natalie

2014-01-01

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

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

PubMed

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

1994-02-28

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

Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

PubMed

Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

2018-02-23

Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

Phage display of engineered binding proteins.

PubMed

Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John

2014-01-01

In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain

NASA Astrophysics Data System (ADS)

Miller, Miles A.; Moss, Marcia L.; Powell, Gary; Petrovich, Robert; Edwards, Lori; Meyer, Aaron S.; Griffith, Linda G.; Lauffenburger, Douglas A.

2015-10-01

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids

NASA Astrophysics Data System (ADS)

Santoshi, Seneha; Naik, Pradeep K.

2014-07-01

Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of -6.4, -7.2, -7.4 and -7.3 kcal/mol with αβI, αβII, αβIII and αβIV isotypes, respectively. Similarly 10 showed higher binding affinity of -6.8 kcal/mol with αβV, whereas 8 had the highest binding affinity of -7.2, -7.1 and -7.2 kcal/mol, respectively with αβVI, αβVII and αβVIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of -46.2 and -38.1 kcal/mol against αβIII (overexpression of αβIII has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.

Two-phase positive inotropic effects of ouabain and the presence of multiple classes of ouabain binding sites in the ferret heart

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

Ng, Y.C.; Akera, T.

1986-03-05

Characteristics of more than one class of ouabain receptors which appear to exist in ferret heart were examined. In isolated papillary muscle, 1 to 30 nM ouabain produced a positive inotropic effect in the presence of 5 ..mu..M propranolol and 2 ..mu..M phentolamine. Higher concentrations of ouabain (0.1 to 10 ..mu..M) produced an additional and prominent inotropic effect. In partially purified Na, K-ATPase, ouabain caused a monophasic inhibition; however, the concentration-inhibition curve spanned over 5 log units, indicating that ouabain is interacting with more than a single class of the enzyme. Scatchard analysis of specific /sup 3/H-ouabain binding revealed approximatelymore » equal abundance of high and low affinity binding sites. The K/sub D/ value for high affinity sites was approximately 20 nM whereas that for low affinity sites was about 45 times higher. When phosphoenzyme was formed in the presence of (..gamma..-/sup 32/P)-ATP, Mg/sup 2 +/ and Na/sup +/ and subjected to SDS gel electrophoresis, two distinct K/sup +/-sensitive bands with about 100,000 dalton molecular weight were detected. Molecular weight difference between these two bands was approximately 2500 dalton. Phosphorylation of either band was abolished by 1 ..mu..M ouabain suggesting that both bands may correspond to the high-affinity binding sites. These results indicate that high and low affinity ouabain binding sites exists in approximately equal abundance in the ferret heart, and that binding of ouabain to these sites cases Na,K-ATPase inhibition and the positive inotropic effect.« less

Sequence2Vec: a novel embedding approach for modeling transcription factor binding affinity landscape.

PubMed

Dai, Hanjun; Umarov, Ramzan; Kuwahara, Hiroyuki; Li, Yu; Song, Le; Gao, Xin

2017-11-15

An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these hidden Markov models into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA datasets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods. Our program is freely available at https://github.com/ramzan1990/sequence2vec. xin.gao@kaust.edu.sa or lsong@cc.gatech.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface.

PubMed

Gorelik, Maryna; Davidson, Alan R

2012-03-16

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

Characterization of the Binding of a Potent Synthetic Androgen, Methyltrienolone, to Human Tissues

PubMed Central

Menon, Mani; Tananis, Catherine E.; Hicks, L. Louise; Hawkins, Edward F.; McLoughlin, Martin G.; Walsh, Patrick C.

1978-01-01

The potent synthetic androgen methytrienolone (R 1881), which does not bind to serum proteins, was utilized to characterize binding to receptors in human androgen responsive tissues. Cytosol extracts prepared from hypertrophic prostates (BPH) were utilized as the source of receptor for the initial studies. High affinity binding was detected in the cytosol of 29 of 30 samples of BPH (average number of binding sites, 45.8±4.7 fmol/mg of protein; dissociation constant, 0.9±0.2 nM). This binding had the characteristics of a receptor: heat lability, precipitability by 0-33% ammonium sulfate and by protamine sulfate, and 8S sedimentation coefficient. High affinity binding was also detected in cytosol prepared from seminal vesicle, epididymis, and genital skin but not in non-genital skin or muscle. However, similar binding was demonstrated in the cytosol of human uterus. The steroid specificities of binding to the cytosol of male tissues of accessory reproduction and of uterus were similar in that progestational agents were more effective competitors than natural androgens. Binding specificities in cytosol prepared from genital skin were distinctly different and were similar to those of ventral prostate from the castrated rat in that dihydrotestosterone was much more potent than progestins in competition. Thus binding of R 1881 to the cytosol of prostate, epididymis, and seminal vesicle has some characteristics of binding to a progesterone receptor. When the nuclear extract from BPH was analyzed, high affinity binding was demonstrated that conformed to the specificities of binding to an androgen receptor. Here dihydrotestosterone was a more potent competitor than progestational agents. Similar patterns of binding were detected in the crude nuclear extracts from seminal vesicle, epididymis, and genital skin but not in uterus, muscle, or non-genital skin. We conclude that the androgen receptor is not demonstrable in the cytosol of prostate, epididymis, or seminal vesicle of non-castrated men but can be measured in the cytosol of genital skin and the nuclear extracts of androgen responsive tissues. Because steroid hormones exert their major influence within the nucleus of target tissues, the measurement of nuclear receptor may provide valuable insight into the regulation of growth of target tissues. PMID:73547

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.

Accurate and sensitive quantification of protein-DNA binding affinity.

PubMed

Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

2018-04-17

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

Accurate and sensitive quantification of protein-DNA binding affinity

PubMed Central

Rastogi, Chaitanya; Rube, H. Tomas; Kribelbauer, Judith F.; Crocker, Justin; Loker, Ryan E.; Martini, Gabriella D.; Laptenko, Oleg; Freed-Pastor, William A.; Prives, Carol; Stern, David L.; Mann, Richard S.; Bussemaker, Harmen J.

2018-01-01

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. PMID:29610332

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

PubMed Central

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

2011-01-01

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

Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders

PubMed Central

Cohen-Khait, Ruth; Schreiber, Gideon

2016-01-01

Protein–protein interactions occur via well-defined interfaces on the protein surface. Whereas the location of homologous interfaces is conserved, their composition varies, suggesting that multiple solutions may support high-affinity binding. In this study, we examined the plasticity of the interface of TEM1 β-lactamase with its protein inhibitor BLIP by low-stringency selection of a random TEM1 library using yeast surface display. Our results show that most interfacial residues could be mutated without a loss in binding affinity, protein stability, or enzymatic activity, suggesting plasticity in the interface composition supporting high-affinity binding. Interestingly, many of the selected mutations promoted faster association. Further selection for faster binders was achieved by drastically decreasing the library–ligand incubation time to 30 s. Preequilibrium selection as suggested here is a novel methodology for specifically selecting faster-associating protein complexes. PMID:27956635

Characterization and autoradiographic localization of neurotensin binding sites in human sigmoid colon.

PubMed

Azriel, Y; Burcher, E

2001-06-01

Radioiodinated neurotensin ((125)I-NT) was used to characterize and localize NT binding sites in normal human sigmoid colon. Specimens were obtained from patients (30-77 years old) undergoing resection for colon carcinoma. Specific binding of (125)I-NT to sigmoid circular muscle membranes was enhanced by o-phenanthroline (1 mM) but other peptidase inhibitors were ineffective. (125)I-NT bound to a high-affinity site of K(d) = 0.88 +/- 0.09 nM and B(max) = 4.03 +/- 0.66 fmol/mg of wet weight tissue (n = 14), although in the majority of patients another site, of low but variable affinity, could also be detected. Specific binding of 50 pM (125)I-NT was inhibited by NT(8-13) > NT > SR142948A > or = neuromedin N > or = SR48692, consistent with binding to the NT1 receptor. In autoradiographic studies, dense specific binding of (125)I-NT was seen over myenteric and submucosal ganglia, moderate binding over circular muscle, and sparse binding over longitudinal muscle and taenia coli. Levocabastine, which has affinity for the NT2 receptor, did not inhibit specific binding of (125)I-NT in membrane competition or autoradiographic studies. NT contracted sigmoid colon circular muscle strips with a pD(2) value of 6.8 +/- 0.2 nM (n = 25). The contractile responses to NT were significantly potentiated in the presence of tetrodotoxin (1 microM), indicating a neural component. Results from functional studies support actions for NT on both muscle and enteric neurons, consistent with the presence of NT receptors on circular muscle and ganglia of human sigmoid colon. The lack of inhibition by levocabastine suggests that the second binding site detected does not correspond to the NT2 receptor.

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.

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)

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

PubMed Central

Will, Katrin; Warnecke, Gabriele; Wiesmüller, Lisa; Deppert, Wolfgang

1998-01-01

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA. PMID:9811860

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Specific DNA binding of the two chicken Deformed family homeodomain proteins, Chox-1.4 and Chox-a.

PubMed Central

Sasaki, H; Yokoyama, E; Kuroiwa, A

1990-01-01

The cDNA clones encoding two chicken Deformed (Dfd) family homeobox containing genes Chox-1.4 and Chox-a were isolated. Comparison of their amino acid sequences with another chicken Dfd family homeodomain protein and with those of mouse homologues revealed that strong homologies are located in the amino terminal regions and around the homeodomains. Although homologies in other regions were relatively low, some short conserved sequences were also identified. E. coli-made full length proteins were purified and used for the production of specific antibodies and for DNA binding studies. The binding profiles of these proteins to the 5'-leader and 5'-upstream sequences of Chox-1.4 and Chox-a coding regions were analyzed by immunoprecipitation and DNase I footprint assays. These two Chox proteins bound to the same sites in the 5'-flanking sequences of their coding regions with various affinities and their binding affinities to each site were nearly the same. The consensus sequences of the high and low affinity binding sites were TAATGA(C/G) and CTAATTTT, respectively. A clustered binding site was identified in the 5'-upstream of the Chox-a gene, suggesting that this clustered binding site works as a cis-regulatory element for auto- and/or cross-regulation of Chox-a gene expression. Images PMID:1970866

Prostaglandin E/sub 2/ localization and receptor identification within the developing murine secondary palate

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

Jones, J.

1986-01-01

Transient elevations in murine secondary palatal adenosine 3',5'-monophosphate (cAMP) levels occur during palate ontogeny. Since palatal processes exposed to dibutyryl cAMP differentiate precociously, increases in palatal cAMP levels are of interest. Prostaglandin E/sub 2/ (PGE/sub 2/), which is synthesized by murine embryonic palate mesenchyme cells (MEPM), regulates cAMP levels in adult tissues via specific membrane bound receptors coupled to adenylate cyclase. Therefore, a PGE/sub 2/ receptor-adenylate cyclase systems was proposed in the developing murine secondary palate. Utilizing a radioligand binding assay, it was determined that murine palatal tissue on day 13 of gestation contained PGE/sub 2/ receptors that were saturable,more » of high affinity and low capacity. Specific (/sup 3/H)-PGE/sub 2/ binding was reversible by 30 min. The order of prostanoid binding affinity at specific PGE/sub 2/ binding sites was E/sub 2/ > F/sub 2//sub ..cap alpha../ > A/sub 2/ > E/sub 1/ = D/sub 2/ indicating specificity of the receptor for PGE/sub 2/. The ability of MEPM cells to respond to PGE/sub 2/ with dose-dependent accumulations of intracellular cAMP demonstrated the functional nature of these binding sites. Analysis of palatal PGE/sub 2/ receptor characteristics on days 12 and 14 of palate development indicated temporal alterations in receptor affinity and density during palate ontogeny.« less

Enriching peptide libraries for binding affinity and specificity through computationally directed library design

PubMed Central

Foight, Glenna Wink; Chen, T. Scott; Richman, Daniel; Keating, Amy E.

2017-01-01

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model. PMID:28236241

Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design.

PubMed

Foight, Glenna Wink; Chen, T Scott; Richman, Daniel; Keating, Amy E

2017-01-01

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.

[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.

Development of Single-Stranded DNA Aptamers for Specific Bisphenol A Detection

PubMed Central

Jo, Minjoung; Ahn, Ji-Young; Lee, Joohyung; Lee, Seram; Hong, Sun Woo; Yoo, Jae-Wook; Kang, Jeehye; Dua, Pooja

2011-01-01

The development of reagents with high affinity and specificity to small molecules is crucial for the high-throughput detection of chemical compounds, such as toxicants or pollutants. Aptamers are short and single-stranded (ss) oligonucleotides able to recognize target molecules with high affinity. Here, we report the selection of ssDNA aptamers that bind to Bisphenol A (BPA), an environmental hormone. Using SELEX process, we isolated high affinity aptamers to BPA from a 1015 random library of 60 mer ssDNAs. The selected aptamers bound specifically to BPA, but not to structurally similar molecules, such as Bisphenol B with one methyl group difference, or 4,4′-Bisphenol with 2 methyl groups difference. Using these aptamers, we developed an aptamer-based sol–gel biochip and detected BPA dissolved in water. This novel BPA aptamer-based detection can be further applied to the universal and high-specificity detection of small molecules. PMID:21413891

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.

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

PubMed

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

1991-07-16

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

Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

PubMed

Nallapareddy, Sreedhar R; Sillanpää, Jouko; Ganesh, Vannakambadi K; Höök, Magnus; Murray, Barbara E

2007-06-01

Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P < 0.0001). Blocking Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

“Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

PubMed Central

Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

2015-01-01

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy. PMID:25837251

Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

PubMed

Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

2018-05-22

Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

Polyadenylation proteins CstF-64 and τCstF-64 exhibit differential binding affinities for RNA polymers

PubMed Central

Monarez, Roberto R.; Macdonald, Clinton C.; Dass, Brinda

2006-01-01

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, τCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between τCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of τCstF-64 and CstF-64 have different affinities for RNA elements. We quantified Kd values of CstF-64 and τCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than τCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal α-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is important in RNA sequence recognition. This supports the hypothesis that τCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements. PMID:17029590

Nucleotide-dependent bisANS binding to tubulin.

PubMed

Chakraborty, S; Sarkar, N; Bhattacharyya, B

1999-07-13

Non-covalent hydrophobic probes such as 5, 5'-bis(8-anilino-1-naphthalenesulfonate) (bisANS) have become increasingly popular to gain information about protein structure and conformation. However, there are limitations as bisANS binds non-specifically at multiple sites of many proteins. Successful use of this probe depends upon the development of binding conditions where only specific dye-protein interaction will occur. In this report, we have shown that the binding of bisANS to tubulin occurs instantaneously, specifically at one high affinity site when 1 mM guanosine 5'-triphosphate (GTP) is included in the reaction medium. Substantial portions of protein secondary structure and colchicine binding activity of tubulin are lost upon bisANS binding in absence of GTP. BisANS binding increases with time and occurs at multiple sites in the absence of GTP. Like GTP, other analogs, guanosine 5'-diphosphate, guanosine 5'-monophosphate and adenosine 5'-triphosphate, also displace bisANS from the lower affinity sites of tubulin. We believe that these multiple binding sites are generated due to the bisANS-induced structural changes on tubulin and the presence of GTP and other nucleotides protect those structural changes.

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

PubMed

Morea, Edna G O; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Matioli, Fabio F; Lira, Cristina B B; Fernandez, Maribel F; Moraes, Barbara S; da Silva, Marcelo S; Storti, Camila B; Fontes, Marcos R M; Cano, Maria Isabel N

2017-11-01

Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. Copyright © 2017 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 Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

PubMed Central

Jiang, Longguang; Paaske, Berit; Kromann-Hansen, Tobias; Jensen, Jan K.; Sørensen, Hans Peter; Liu, Zhuo; Nielsen, Jakob T.; Christensen, Anni; Hosseini, Masood; Sørensen, Kasper K.; Nielsen, Niels Christian; Jensen, Knud J.; Huang, Mingdong; Andreasen, Peter A.

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden. PMID:25545505

Production and characterization of a high-affinity nanobody against human endoglin.

PubMed

Ahmadvand, Davoud; Rasaee, Mohammad J; Rahbarizadeh, Fatemeh; Mohammadi, Mohammad

2008-10-01

Abstract Antibodies or antibody fragments are almost exclusively applied in human therapy and diagnosis. The high affinity and specificity of antibodies makes them suitable for these applications. Nanobody, the variable domain of Camelidae heavy chain antibodies, have superior properties compared with conventional antibodies in that they are small, non-immunogenic, very stable, highly soluble, and easy to produce in large quantities. In the present study, we report the isolation and characterization of a high-affinity binder against human endoglin retrieved from camels' nanobody gene library. Endoglin (CD105), an accessory protein of the transforming growth factor beta receptor complex, has become an attractive molecule for the targeting of the tumor vasculature. Upregulation of endoglin on proliferating endothelial cells is associated with tumor neovascularization. Here, we generated two nanobody gene libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the recombinant extracellular domain of human endoglin. The other selected anti-endoglin nanobody (AR1-86) showed strong binding to human endoglin expressing endothelial cells (HUVECs), while no binding was observed with the endoglin-negative cell line (HEK293). This high-affinity single-domain antibody could be a good candidate for the generation of vascular or tumor targeting agents in cancer therapy.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

PubMed

Inglis, Steven R; Stojkoski, Cvetan; Branson, Kim M; Cawthray, Jacquie F; Fritz, Daniel; Wiadrowski, Emma; Pyke, Simon M; Booker, Grant W

2004-10-21

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

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.

[(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

PubMed

Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

2002-02-11

This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

Molecular principle of the cyclin-dependent kinase selectivity of 4-(thiazol-5-yl)-2-(phenylamino) pyrimidine-5-carbonitrile derivatives revealed by molecular modeling studies.

PubMed

Kong, Xiaotian; Sun, Huiyong; Pan, Peichen; Tian, Sheng; Li, Dan; Li, Youyong; Hou, Tingjun

2016-01-21

Due to the high sequence identity of the binding pockets of cyclin-dependent kinases (CDKs), designing highly selective inhibitors towards a specific CDK member remains a big challenge. 4-(thiazol-5-yl)-2-(phenylamino) pyrimidine derivatives are effective inhibitors of CDKs, among which the most promising inhibitor 12u demonstrates high binding affinity to CDK9 and attenuated binding affinity to other homologous kinases, such as CDK2. In this study, in order to rationalize the principle of the binding preference towards CDK9 over CDK2 and to explore crucial information that may aid the design of selective CDK9 inhibitors, MM/GBSA calculations based on conventional molecular dynamics (MD) simulations and enhanced sampling simulations (umbrella sampling and steered MD simulations) were carried out on two representative derivatives (12u and 4). The calculation results show that the binding specificity of 12u to CDK9 is primarily controlled by conformational change of the G-loop and variation of the van der Waals interactions. Furthermore, the enhanced sampling simulations revealed the different reaction coordinates and transient interactions of inhibitors 12u and 4 as they dissociate from the binding pockets of CDK9 and CDK2. The physical principles obtained from this study may facilitate the discovery and rational design of novel and specific inhibitors of CDK9.

The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

PubMed Central

Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

1992-01-01

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates.

PubMed

Deeg, Andreas A; Reiner, Anne M; Schmidt, Felix; Schueder, Florian; Ryazanov, Sergey; Ruf, Viktoria C; Giller, Karin; Becker, Stefan; Leonov, Andrei; Griesinger, Christian; Giese, Armin; Zinth, Wolfgang

2015-09-01

Special diphenyl-pyrazole compounds and in particular anle138b were found to reduce the progression of prion and Parkinson's disease in animal models. The therapeutic impact of these compounds was attributed to the modulation of α-synuclein and prion-protein aggregation related to these diseases. Photophysical and photochemical properties of the diphenyl-pyrazole compounds anle138b, anle186b and sery313b and their interaction with monomeric and aggregated α-synuclein were studied by fluorescence techniques. The fluorescence emission of diphenyl-pyrazole is strongly increased upon incubation with α-synuclein fibrils, while no change in fluorescence emission is found when brought in contact with monomeric α-synuclein. This points to a distinct interaction between diphenyl-pyrazole and the fibrillar structure with a high binding affinity (Kd=190±120nM) for anle138b. Several α-synuclein proteins form a hydrophobic binding pocket for the diphenyl-pyrazole compound. A UV-induced dehalogenation reaction was observed for anle138b which is modulated by the hydrophobic environment of the fibrils. Fluorescence of the investigated diphenyl-pyrazole compounds strongly increases upon binding to fibrillar α-synuclein structures. Binding at high affinity occurs to hydrophobic pockets in the fibrils. The observed particular fluorescence properties of the diphenyl-pyrazole molecules open new possibilities for the investigation of the mode of action of these compounds in neurodegenerative diseases. The high binding affinity to aggregates and the strong increase in fluorescence upon binding make the compounds promising fluorescence markers for the analysis of aggregation-dependent epitopes. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

PubMed

Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

2014-04-01

The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

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

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE PAGES

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

2015-12-18

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

The development and application of a quantitative peptide microarray platform to SH2 domain specificity space

NASA Astrophysics Data System (ADS)

Engelmann, Brett Warren

The Src homology 2 (SH2) domains evolved alongside protein tyrosine kinases (PTKs) and phosphatases (PTPs) in metazoans to recognize the phosphotyrosine (pY) post-translational modification. The human genome encodes 121 SH2 domains within 111 SH2 domain containing proteins that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. Despite pY recognition contributing to roughly half of the binding energy, SH2 domains possess substantial binding specificity, or affinity discrimination between phosphopeptide ligands. This specificity is largely imparted by amino acids (AAs) adjacent to the pY, typically from positions +1 to +4 C-terminal to the pY. Much experimental effort has been undertaken to construct preferred binding motifs for many SH2 domains. However, due to limitations in previous experimental methodologies these motifs do not account for the interplay between AAs. It was therefore not known how AAs within the context of individual peptides function to impart SH2 domain specificity. In this work we identified the critical role context plays in defining SH2 domain specificity for physiological ligands. We also constructed a high quality interactome using 50 SH2 domains and 192 physiological ligands. We next developed a quantitative high-throughput (Q-HTP) peptide microarray platform to assess the affinities four SH2 domains have for 124 physiological ligands. We demonstrated the superior characteristics of our platform relative to preceding approaches and validated our results using established biophysical techniques, literature corroboration, and predictive algorithms. The quantitative information provided by the arrays was leveraged to investigate SH2 domain binding distributions and identify points of binding overlap. Our microarray derived affinity estimates were integrated to produce quantitative interaction motifs capable of predicting interactions. Furthermore, our microarrays proved capable of resolving subtle contextual differences within motifs that modulate interaction affinities. We conclude that contextually informed specificity profiling of protein interaction domains using the methodologies developed in this study can inform efforts to understand the interconnectivity of signaling networks in normal and aberrant states. Three supplementary tables containing detailed lists of peptides, interactions, and sources of corroborative information are provided.

Targeted binding of the M13 bacteriophage to thiamethoxam organic crystals.

PubMed

Cho, Whirang; Fowler, Jeffrey D; Furst, Eric M

2012-04-10

Phage display screening with a combinatorial library was used to identify M13-type bacteriophages that express peptides with selective binding to organic crystals of thiamethoxam. The six most strongly binding phages exhibit at least 1000 times the binding affinity of wild-type M13 and express heptapeptide sequences that are rich in hydrophobic, hydrogen-bonding amino acids and proline. Among the peptide sequences identified, M13 displaying the pIII domain heptapeptide ASTLPKA exhibits the strongest binding to thiamethoxam in competitive binding assays. Electron and confocal microscopy confirm the specific binding affinity of ASTLPKA to thiamethoxam. Using atomic force microscope (AFM) probes functionalized with ASTLPKA expressing phage, we found that the average adhesion force between the bacteriophage and a thiamethoxam surface is 1.47 ± 0.80 nN whereas the adhesion force of wild-type M13KE phage is 0.18 ± 0.07 nN. Such a strongly binding bacteriophage could be used to modify the surface chemistry of thiamethoxam crystals and other organic solids with a high degree of specificity. © 2012 American Chemical Society

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.

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

PubMed Central

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

2010-01-01

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

High-aluminum-affinity silica is a nanoparticle that seeds secondary aluminosilicate formation.

PubMed

Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J

2013-01-01

Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m(2) g(-1) and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates.

High-Aluminum-Affinity Silica Is a Nanoparticle That Seeds Secondary Aluminosilicate Formation

PubMed Central

Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J.

2013-01-01

Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m2 g-1 and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates. PMID:24349573

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

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

Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.

2011-02-01

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

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

Structure of dual receptor binding to botulinum neurotoxin B.

PubMed

Berntsson, Ronnie P-A; Peng, Lisheng; Dong, Min; Stenmark, Pål

2013-01-01

Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3-Å structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points.

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process.

PubMed

Kirchberg, Kristina; Kim, Tai-Yang; Möller, Martina; Skegro, Darko; Dasara Raju, Gayathri; Granzin, Joachim; Büldt, Georg; Schlesinger, Ramona; Alexiev, Ulrike

2011-11-15

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process

PubMed Central

Kirchberg, Kristina; Kim, Tai-Yang; Möller, Martina; Skegro, Darko; Dasara Raju, Gayathri; Granzin, Joachim; Büldt, Georg; Schlesinger, Ramona; Alexiev, Ulrike

2011-01-01

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the “phosphate-sensor” of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins. PMID:22039220

Miniature protein ligands for EVH1 domains: Interplay between affinity, specificity, and cell motility⊥

PubMed Central

Holtzman, Jennifer H.; Woronowicz, Kamil; Golemi-Kotra, Dasantila; Schepartz, Alanna

2008-01-01

Dynamic rearrangements of the actin cytoskeleton power cell motility in contexts ranging from intracellular microbial pathogenesis to axon guidance. The Ena/VASP family proteins--Mena, VASP, and Evl--are believed to control cell motility by serving as a direct link between signaling events and the actin cytoskeleton. Our lab has previously reported a novel miniature protein, pGolemi, which binds with high affinity to the EVH1 domain of Mena (Mena1-112) but not to those of VASP (VASP1-115) or Evl (Evl1-115) and also causes an unusual defect in actin-driven L. monocytogenes motility. Here, we use scanning mutagenesis to examine the effects of single amino acid changes within pGolemi on EVH1 domain affinity and specificity, miniature protein secondary structure, and L. monocytogenes motility. The data suggest that pGolemi contains the expected aPP-like fold and binds Mena1-112 in a manner highly analogous to the proline-rich repeat region of L. monocytogenes ActA protein. Residues throughout pGolemi contribute to both EVH1 domain affinity and paralog specificity. Moreover, the affinities of pGolemi variants for Mena1-112 correlate with selectivity against the EVH1 domains of VASP and Evl. In L. monocytogenes motility assays, speed and speed variability correlate strongly with EVH1 paralog specificity, suggesting that the Ena/VASP paralogs do not play equivalent roles in the process of L. monocytogenes actin tail maturation. PMID:17973491

Computational Analysis of Sterol Ligand Specificity of the Niemann Pick C2 Protein.

PubMed

Poongavanam, Vasanthanathan; Kongsted, Jacob; Wüstner, Daniel

2016-09-13

Transport of cholesterol derived from hydrolysis of lipoprotein associated cholesteryl esters out of late endosomes depends critically on the function of the Niemann Pick C1 (NPC1) and C2 (NPC2) proteins. Both proteins bind cholesterol but also various other sterols and both with strongly varying affinity. The molecular mechanisms underlying this multiligand specificity are not known. On the basis of the crystal structure of NPC2, we have here investigated structural details of NPC2-sterol interactions using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations. We found that an aliphatic side chain in the sterol ligand results in strong binding to NPC2, while side-chain oxidized sterols gave weaker binding. Estradiol and the hydrophobic amine U18666A had the lowest affinity of all tested ligands and at the same time showed the highest flexibility within the NPC2 binding pocket. The binding affinity of all ligands correlated highly with their calculated partitioning coefficient (logP) between octanol/water phases and with the potential of sterols to stabilize the protein backbone. From molecular dynamics simulations, we suggest a general mechanism for NPC2 mediated sterol transfer, in which Phe66, Val96, and Tyr100 act as reversible gate keepers. These residues stabilize the sterol in the binding pose via π-π stacking but move transiently apart during sterol release. A computational mutation analysis revealed that the binding of various ligands depends critically on the same specific amino acid residues within the binding pocket providing shape complementary to sterols, but also on residues in distal regions of the protein.

Modulation of DNA binding by gene-specific transcription factors.

PubMed

Schleif, Robert F

2013-10-01

The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

PubMed Central

Owczarek, C M; Layton, M J; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

1993-01-01

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins. Images PMID:8253075

Plasma sex steroid binding in Chiroptera.

PubMed

Kwiecinski, G G; Damassa, D A; Gustafson, A W; Armao, M E

1987-04-01

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.

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

Mechanically tunable actin networks using programmable DNA based cross-linkers

NASA Astrophysics Data System (ADS)

Schnauss, Joerg; Lorenz, Jessica; Schuldt, Carsten; Kaes, Josef; Smith, David

Cells employ multiple cross-linkers with very different properties. Studies of the entire phase space, however, were infeasible since they were restricted to naturally occurring cross-linkers. These components cannot be controllably varied and differ in many parameters. We resolve this limitation by forming artificial actin cross-linkers, which can be controllably varied. The basic building block is DNA enabling a well-defined length variation. DNA can be attached to actin binding peptides with known binding affinities. We used bulk rheology to investigate mechanical properties of these networks. We were able to reproduce mechanical features of actin networks cross-linked by fascin by using a short version of our artificial complex with a high binding affinity. Additionally, we were able to resemble findings for the cross-linker alpha-actinin by employing a long cross-linker with a low binding affinity. Between these natural limits we investigated three different cross-linker lengths each with two different binding affinities. With these controlled variations we are able to precisely screen the phase space of cross-linked actin networks by changing only one specific parameter and not the entire set of properties as in the case of naturally occurring cross-linking complexes.

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

PubMed

Yandell, C A; Dunbar, A J; Wheldrake, J F; Upton, Z

1999-09-17

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

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

Klein, M.; Canoll, P.D.; Musacchio, J.M.

1991-01-01

The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drugmore » has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.« less

Recombinant phage probes for Listeria monocytogenes

NASA Astrophysics Data System (ADS)

Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

2007-10-01

Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Modulating the DNA affinity of Elk-1 with computationally selected mutations.

PubMed

Park, Sheldon; Boder, Eric T; Saven, Jeffery G

2005-04-22

In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

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

PubMed Central

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

2015-01-01

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

Insights into Protein–Ligand Interactions: Mechanisms, Models, and Methods

PubMed Central

Du, Xing; Li, Yi; Xia, Yuan-Ling; Ai, Shi-Meng; Liang, Jing; Sang, Peng; Ji, Xing-Lai; Liu, Shu-Qun

2016-01-01

Molecular recognition, which is the process of biological macromolecules interacting with each other or various small molecules with a high specificity and affinity to form a specific complex, constitutes the basis of all processes in living organisms. Proteins, an important class of biological macromolecules, realize their functions through binding to themselves or other molecules. A detailed understanding of the protein–ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein-ligand recognition and binding will also facilitate the discovery, design, and development of drugs. In the present review, first, the physicochemical mechanisms underlying protein–ligand binding, including the binding kinetics, thermodynamic concepts and relationships, and binding driving forces, are introduced and rationalized. Next, three currently existing protein-ligand binding models—the “lock-and-key”, “induced fit”, and “conformational selection”—are described and their underlying thermodynamic mechanisms are discussed. Finally, the methods available for investigating protein–ligand binding affinity, including experimental and theoretical/computational approaches, are introduced, and their advantages, disadvantages, and challenges are discussed. PMID:26821017

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

PubMed

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

2016-10-01

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

A low-complexity region in the YTH domain protein Mmi1 enhances RNA binding.

PubMed

Stowell, James A W; Wagstaff, Jane L; Hill, Chris H; Yu, Minmin; McLaughlin, Stephen H; Freund, Stefan M V; Passmore, Lori A

2018-06-15

Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR, and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1-RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA-binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding. © 2018 Stowell et al.

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

PubMed

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

2016-05-01

Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Rapid characterization of a novel taspine derivative-HMQ1611 binding to EGFR by a cell membrane chromatography method.

PubMed

Du, Hui; Lv, Nan; Wang, Sicen; He, Langchong

2013-05-01

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor's concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (K(D)) was (9.11 ± 1.89) × 10(-6) M; HMQ1611 had two major binding regions on the EGFR, and the K(D) values obtained from the model were (2.39 ± 0.33) × 10(-7) and (3.87 ± 0.93) × 10(-5) M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

PubMed

Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

1995-09-05

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

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

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

NASA Astrophysics Data System (ADS)

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

2016-04-01

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

ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

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

HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5more » (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).« less

A Single Glycine-Alanine Exchange Directs Ligand Specificity of the Elephant Progestin Receptor

PubMed Central

Wierer, Michael; Schrey, Anna K.; Kühne, Ronald; Ulbrich, Susanne E.

2012-01-01

The primary gestagen of elephants is 5α-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems. PMID:23209719

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

PubMed

Nuttall, Stewart D; Krishnan, Usha V; Doughty, Larissa; Pearson, Kylie; Ryan, Michael T; Hoogenraad, Nicholas J; Hattarki, Meghan; Carmichael, Jennifer A; Irving, Robert A; Hudson, Peter J

2003-09-01

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

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

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

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.

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.

Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

PubMed

Liu, Mingming; Huang, Rong; Weisman, Adam; Yu, Xiaoyang; Lee, Shih-Hui; Chen, Yalu; Huang, Chao; Hu, Senhua; Chen, Xiuhua; Tan, Wenfeng; Liu, Fan; Chen, Hao; Shea, Kenneth J

2018-05-24

We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R 1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280 FRGSAQGIEGS 290 , 368 RRPFNIGINNQQ 379 and 436 FRSGFSNSSVSIIR 449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368 RRPFNIGINNQQ 379 . This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865 NITIHITDTNNK 876 , a specific recognition site in insect receptor Bt-R 1 , to 368 RRPFNIGINNQQ 379 . The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368 RRPFNIGINNQQ 379 . This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

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

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

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

PubMed

De Marco, L; Mazzucato, M; Masotti, A; Ruggeri, Z M

1994-03-04

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa.

PubMed

Wylie, J L; Worobec, E A

1993-07-01

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.

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

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva† †Electronic supplementary information (ESI) available: Optimization of Mg2+ and ATMND concentrations for our CBSA-based ATMND-binding assay; ATMND-reported calibration curve for CBSA-5325 at various cocaine concentrations; ATMND binding affinity for the cocaine-assembled CBSA-5325; K D of 38-GC and different 38-GC mutants for cocaine as characterized by ITC; stem length effects on cocaine-induced CBSA assembly; spectra of CBSA-5335-based fluorescence detection of cocaine in 1× binding buffer; characterization of cocaine binding affinity of CBSA-5335 and PSA using ITC; fluorescence detection of cocaine in saliva with our fluorophore/quencher modified CBSA-5335; calibration curve of our CBSA-5335-based fluorophore/quencher assay in 1× binding buffer and 10% saliva at cocaine concentrations ranging from 0 to 10 μM; bias and precision of the CBSA-5335-based fluorophore/quencher assay; comparison of amplification-free split-aptamer assays for cocaine detection; sequence ID and DNA sequences used in this work. See DOI: 10.1039/c6sc01833e Click here for additional data file.

PubMed Central

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui

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. PMID:28451157

Interaction of xenobiotics with estrogen receptors α and β and a putative plasma sex hormone-binding globulin from channel catfish (Ictalurus punctatus)

USGS Publications Warehouse

Gale, William L.; Patino, Reynaldo; Maule, Alec G.

2004-01-01

Estrogens are important regulators of physiological functions. Although environmental contaminants (xenoestrogens) which interfere with estrogen signaling are of increasing concern, there is only limited information about their ability to interact with estrogen-binding proteins (SHBG) or receptors (ER). Recombinant ER?? and ?? were obtained after transient transfection of COS-7 cells with channel catfish ER cDNA. Plasma from adult female channel catfish was the source of SHBG. Tritiated estradiol ( 3H-E2) was used in standard radioligand-binding assays to characterize the binding properties of channel catfish SHBG (ccfSHBG) and to estimate the inhibition constants for various estrogenic compounds. Binding of 3H-E2 to ccfSHBG was saturable and of high affinity with a Kd (??SE) of 1.9??0.14nM and a Bmax of 14.3??2.4pmol/mg protein (n=3 assays). Additionally, ccfSHBG displayed binding specificity for androgens and estrogens. Endosulfan, 4-nonylphenol, and 4-octylphenol displaced 3H-E2 binding to ccfSHBG albeit only at very high concentrations, whereas dieldrin and atrazine showed little displacement activity even at the highest concentrations used. The synthetic estrogen ethynylestradiol had higher affinity than E2 for ccfSHBG. This finding differs from results with human and rainbow trout SHBG. The alkylphenolic compounds (4-octylphenol and 4-nonylphenol) displayed some ability to displace 3H-E2 binding from ER?? and ?? at high concentrations, but dieldrin and atrazine had little binding activity for both ER subtypes and endosulfan for ER??. The xenobiotics tested generally showed equivalent or greater affinity for ER?? than ER??, whereas natural estrogens had much greater affinity for ER?? than ER??. These observations suggest that results of studies using fish tissue ER extracts must be interpreted with caution, since both ER subtypes may be present, and that the binding of xenoestrogens to SHBG must be taken into account for proper assessment of endocrine disruption caused by environmental contaminants.

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

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

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.

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

PubMed

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

2006-03-23

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

Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

PubMed

Raj, Isha; Sadat Al Hosseini, Hamed; Dioguardi, Elisa; Nishimura, Kaoru; Han, Ling; Villa, Alessandra; de Sanctis, Daniele; Jovine, Luca

2017-06-15

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cortisol-21-sulfate (FS) is a specific ligand for intracellular transcortin: demonstration of three types of high affinity corticosteroid binders in bovine aortic cytosol by a combined use of FS and RU 28362.

PubMed

Hayashi, T; Kornel, L

1990-01-01

This paper reports the results of a study on the binding of adrenal steroids in bovine aortic tissue. Using the same method as in our previous study of mineralocorticoid and glucocorticoid binding in rabbit arterial cytosol, we could not demonstrate in the bovine aorta the three types of high affinity binders for these steroids, which we found in the rabbit arteries. In the search for specific markers for each of the three types of binders (glucocorticoid and mineralocorticoid receptors and the transcortin-like intracellular binder), we have found that a conjugated steroid, cortisol-21-sulfate, binds preferentially to the transcortin-like binder, but not to the two receptors. Using this steroid, in combination with the pure synthetic glucocorticoid RU 28362, we were able to clearly discriminate between the three types of corticosteroid binders in bovine aorta.

Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

NASA Astrophysics Data System (ADS)

Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

2009-02-01

Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay.

PubMed Central

Prestwich, G D; Wawrzeńczyk, C

1985-01-01

A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites. PMID:3860862

Feature selection and classification of protein-protein complexes based on their binding affinities using machine learning approaches.

PubMed

Yugandhar, K; Gromiha, M Michael

2014-09-01

Protein-protein interactions are intrinsic to virtually every cellular process. Predicting the binding affinity of protein-protein complexes is one of the challenging problems in computational and molecular biology. In this work, we related sequence features of protein-protein complexes with their binding affinities using machine learning approaches. We set up a database of 185 protein-protein complexes for which the interacting pairs are heterodimers and their experimental binding affinities are available. On the other hand, we have developed a set of 610 features from the sequences of protein complexes and utilized Ranker search method, which is the combination of Attribute evaluator and Ranker method for selecting specific features. We have analyzed several machine learning algorithms to discriminate protein-protein complexes into high and low affinity groups based on their Kd values. Our results showed a 10-fold cross-validation accuracy of 76.1% with the combination of nine features using support vector machines. Further, we observed accuracy of 83.3% on an independent test set of 30 complexes. We suggest that our method would serve as an effective tool for identifying the interacting partners in protein-protein interaction networks and human-pathogen interactions based on the strength of interactions. © 2014 Wiley Periodicals, Inc.

Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition

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

Krejciríková, Veronika; Pachl, Petr; Fábry, Milan

2011-11-18

Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 {angstrom} resolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K{sub d} value in the micromolar range (K{submore » d1} = 600 {+-} 70 {mu}M) and a low-affinity site with K{sub d2} = 28 {+-} 10 mM.« less

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

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.

Denervation does not alter the number of neuronal bungarotoxin binding sites on autonomic neurons in the frog cardiac ganglion.

PubMed

Sargent, P B; Bryan, G K; Streichert, L C; Garrett, E N

1991-11-01

The binding of neuronal bungarotoxin (n-BuTX; also known as bungarotoxin 3.1, kappa-bungarotoxin, and toxin F) was analyzed in normal and denervated parasympathetic cardiac ganglia of the frog Rana pipiens, n-BuTX blocks both EPSPs and ACh potentials at 5-20 nM, as determined by intracellular recording techniques. Scatchard analysis on homogenates indicates that cardiac ganglia have two classes of binding sites for 125I-n-BuTX: a high-affinity site with an apparent dissociation constant (Kd,app) of 1.7 nM and a Bmax (number of binding sites) of 3.8 fmol/ganglion and a low-affinity site with a Kd,app of 12 microM and a Bmax of 14 pmol/ganglion. alpha-Bungarotoxin does not appear to interfere with the binding of 125I-n-BuTX to either site. The high-affinity binding site is likely to be the functional nicotinic ACh receptor (AChR), given the similarity between its affinity for 125I-n-BuTX and the concentration of n-BuTX required to block AChR function. Light microscopic autoradiographic analysis of 125I-n-BuTX binding to the ganglion cell surface reveals that toxin binding is concentrated at synaptic sites, which were identified using a synaptic vesicle-specific antibody. Scatchard analysis of autoradiographic data reveals that 125I-n-BuTX binding to the neuronal surface is saturable and has a Kd,app similar to that of the high-affinity binding site characterized in homogenates. Surface binding of 125I-n-BuTX is blocked by nicotine, carbachol, and d-tubocurarine (IC50 less than 20 microM), but not by atropine (IC50 greater than 10 mM). Denervation of the heart increases the ACh sensitivity of cardiac ganglion cells but has no effect upon the number of high-affinity binding sites for 125I-n-BuTX in tissue homogenates. Moreover, autoradiographic analysis indicates that denervation does not alter the number of 125I-n-BuTX binding sites on the ganglion cell surface. n-BuTX is as effective in reducing ganglion cell responses to ACh in denervated ganglia as it is in normally innervated ganglia. These results suggest that denervation alters neither the total number of nicotinic AChRs in the cardiac ganglion nor the number found on the surface of ganglion cells. These autonomic neurons thus respond differently to denervation than do skeletal myofibers. The increase in ACh sensitivity displayed by cardiac ganglion cells upon denervation cannot be explained by changes in AChR number.

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.

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.

Urban Biomining: Biological Extraction of Metals and Materials from Electronics Waste Using a Synthetic Biology Approach

NASA Astrophysics Data System (ADS)

Urbina-Navarrete, J.; Rothschild, L.

2016-12-01

End-of-life electronics waste (e-waste) containing toxic and valuable materials is a rapidly progressing human health and environmental issue. Using synthetic biology tools, we have developed a recycling method for e-waste. Our innovation is to use a recombinant version of a naturally-occurring silica-degrading enzyme to depolymerize the silica in metal- and glass- containing e-waste components, and subsequently, to use engineered bacterial surfaces to bind and separate metals from a solution. The bacteria with bound metals can then be used as "bio-ink" to print new circuits using a novel plasma jet electronics printing technology. Here, we present the results from our initial studies that focus on the specificity of metal-binding motifs for a cognate metal. The candidate motifs that show high affinity and specificity will be engineered into bacterial surfaces for downstream applications in biologically-mediated metal recycling. Since the chemistry and role of Cu in metalloproteins is relatively well-characterized, we are using Cu as a proxy to elucidate metal and biological ligand interactions with various metals in e-waste. We assess the binding parameters of 3 representative classes of Cu-binding motifs using isothermal titration calorimetry; 1) natural motifs found in metalloproteins, 2) consensus motifs, and 3) rationally designed peptides that are predicted, in silico, to bind Cu. Our results indicate that naturally-occurring motifs have relative high affinity and specificity for Cu (association constant for Cu Ka 104 M-1, Zn Ka 103 M-1) when competing ions are present in the aqueous milieu. However, motifs developed through rational design by applying quantum mechanical methods that take into account complexation energies of the elemental binding partners and molecular geometry of the cognate metal, not only show high affinity for the cognate metal (Cu Ka 106 M-1), but they show specificity and discrimination against other metal ions that would be competitors for the same binding sites. This is an initial proof-of-concept study that focuses on Cu-binding; however the overall objective of this research is to have peptides that selectively bind many metals from e-waste and this would allow for the separation of the metals from a solution, at ambient temperatures and under non-toxic conditions.

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

PubMed Central

Vassylyeva, Marina N.; Klyuyev, Sergiy; Vassylyev, Alexey D.; Wesson, Hunter; Zhang, Zhuo; Renfrow, Matthew B.; Wang, Hengbin; Higgins, N. Patrick; Chow, Louise T.; Vassylyev, Dmitry G.

2017-01-01

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10−14–10−17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays. PMID:28607052

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

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

Nye, J.S.

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

[Separation of osteoclasts by lectin affinity chromatography].

PubMed

Itokazu, M; Tan, A; Tanaka, S

1991-09-01

Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach

PubMed Central

Morgan, Hugh P.; Estibeiro, Peter; Wear, Martin A.; Max, Klaas E.A.; Heinemann, Udo; Cubeddu, Liza; Gallagher, Maurice P.; Sadler, Peter J.; Walkinshaw, Malcolm D.

2007-01-01

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function. PMID:17488853

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

Lever, J.R.; Scheffel, U.; Stathis, M.

Analogues of diprenorphine (DPN) having C6-O-iodoallyl (O-IA-DPN) and N-iodoallyl (N-IA-DPN) substituents can be I-125 labeled in good yield with high specific activity by radioiododestannylation. When tested in vitro against [H-3]-DPN in rat brain membranes, the apparent affinity (Ki) of O-IA-DPN (1.35 nM) proved 17-fold stronger than that of N-IA-DPN (23.4 nM). Against selective [H-3]-ligands, O-IA-DPN showed high apparent affinities for {mu}(1.9 nM), {gamma}(1.1 nM) and {kappa}(0.9 nM) sites. Consistent with the low apparent affinity in vitro, [I-125]-N-IA- DPN did not allow localization of cerebral opioid receptors after i.v. administration to mice. By contrast, [I-125]-O-IA-DPN exhibited a regional brain distribution whichmore » reflects binding to multiple opioid receptors. The highest radioactivity concentrations were in superior colliculi, hypothalamus, olfactory tubercles, thalamus and striatum. Peak levels (2.5-3.5 %ID/g) were maintained over the first 60 min. At all times, the lowest levels of radioactivity were in the cerebellum. Binding in vivo was saturable by O-IA-DPN, was blocked by (-)- but not by (+)-naloxone, and was inhibited by naltrexone in dose-dependent fashion. Specific binding was 83-93% for all tissues except cerebellum, where 50% blockade was noted with naltrexone (5.0 mg/kg). Using naltrexone blockade to define non-specific binding, the highest ratio of specific to non-specific binding (> 14 to 1) was noted for superior colliculi at 60 min. Inhibition studies with drugs selective for {mu}, {gamma} or {kappa} sites established that multiple opioid receptors are labeled. [123I]-O-IA-DPN has been prepared (84%, >2400 mCi/{mu}mol), and allows visualization of opioid receptors in mouse brain by ex vivo autoradiography. Together, these results suggest that [123I]-O-IA-DPN is suitable for SPECT studies of multiple opioid receptors.« less

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

PubMed

Tan, Cheng; Li, Wenfei; Wang, Wei

2013-12-19

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Native Electrospray Ionization Mass Spectrometry Reveals Multiple Facets of Aptamer-Ligand Interactions: From Mechanism to Binding Constants.

PubMed

Gülbakan, Basri; Barylyuk, Konstantin; Schneider, Petra; Pillong, Max; Schneider, Gisbert; Zenobi, Renato

2018-06-20

Aptamers are oligonucleotide receptors obtained through an iterative selection process from random-sequence libraries. Though many aptamers for a broad range of targets with high affinity and selectivity have been generated, a lack of high-resolution structural data and the limitations of currently available biophysical tools greatly impede understanding of the mechanisms of aptamer-ligand interactions. Here we demonstrate that an approach based on native electrospray ionization mass spectrometry (ESI-MS) can be successfully applied to characterize aptamer-ligand complexes in all details. We studied an adenosine-binding aptamer (ABA), a l-argininamide-binding aptamer (LABA), and a cocaine-binding aptamer (CBA) and their noncovalent interactions with ligands by native ESI-MS and complemented these measurements by ion mobility spectrometry (IMS), isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. The ligand selectivity of the aptamers and the respective complex stoichiometry could be determined by the native ESI-MS approach. The ESI-MS data can also help refining the binding model for aptamer-ligand complexes and deliver accurate aptamer-ligand binding affinities for specific and nonspecific binding events. For specific ligands, we found K d1 = 69.7 μM and K d2 = 5.3 μM for ABA (two binding sites); K d1 = 22.04 μM for LABA; and K d1 = 8.5 μM for CBA.

AFRRI Reports, Second Quarter 1994

DTIC Science & Technology

1994-08-01

the antrum wete immediately placed in sterile 0.9% NaCl, kept on ice, coded, and then prepared for culture, smears, and urease assay by homogeniza...high urease specific activity (>1 |J.mol- min-1 ■ mg protein-1) plus high-affinity substrate binding (Mi- chaelis constant [K^\\ < 1 mmol/L),27 in at...031, respectively), and the characteristic bacterial growth with high-activity product.on of a urease with tight substrate binding " was found in

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.

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

PubMed

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

2012-04-15

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

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

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.

Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from Dastarcus helophoroides.

PubMed

Yang, Rui-Nan; Li, Dong-Zhen; Yu, Guangqiang; Yi, Shan-Cheng; Zhang, Yinan; Kong, De-Xin; Wang, Man-Qun

2017-12-01

In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-β-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (β-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-β-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, β-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.

Structural insights into Aspergillus fumigatus lectin specificity: AFL binding sites are functionally non-equivalent.

PubMed

Houser, Josef; Komarek, Jan; Cioci, Gianluca; Varrot, Annabelle; Imberty, Anne; Wimmerova, Michaela

2015-03-01

The Aspergillus fumigatus lectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological applications.

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.

Target-molecule-triggered rupture of aptamer-encapsulated polyelectrolyte microcapsules.

PubMed

Zhang, Xueru; Chabot, Denise; Sultan, Yasir; Monreal, Carlos; DeRosa, Maria C

2013-06-26

Polyelectrolyte microcapsules have great potential for serving as carriers for the delivery of their contents when triggered by an external stimulus. Aptamers are synthetic ssDNA or RNA that can bind to specific targets with high affinity and selectivity. Aptamers may retain these superior molecular recognition properties after encapsulation within polymer microcapsules. In this work, stable polyelectrolyte microcapsules with encapsulated aptamers were obtained by the layer-by-layer (LbL) method. Polyelectrolyte films were deposited onto a CaCO3 template that had been predoped with polystyrene sulfonate (PSS) and aptamer sequences (SA) that have an affinity for the dye sulforhodamine B (SRB). The PSS and aptamers are thought to serve as an internal scaffold supporting the microcapsule walls. These microcapsules would present target-molecule-triggered rupture properties. Microcapsule collapse was triggered by the binding of SRB to the encapsulated aptamer. The specificity of microcapsule collapse was investigated using a similar dye, tetramethylrosamine (TMR), which does not have affinity for SA. A high concentration of TMR did not lead to the collapse of the microcapsules. The effect of target binding on the microcapsules was confirmed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). These microcapsules may have potential applications in targeted delivery systems for the controlled release of drugs, pesticides, or other payloads.

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

PubMed

Tian, Z; Zhang, Y

2016-12-01

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

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.

New Horizons on Molecular Pharmacology Applied to Drug Discovery: When Resonance Overcomes Radioligand Binding.

PubMed

Pernomian, Larissa; Gomes, Mayara Santos; Moreira, Josimar Dornelas; da Silva, Carlos Henrique Tomich de Paula; Rosa, Joaquin Maria Campos; Cardoso, Cristina Ribeiro de Barros

2017-01-01

One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Two amino acid residues confer different binding affinities of Abelson family kinase SRC homology 2 domains for phosphorylated cortactin.

PubMed

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

2014-07-11

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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

2015-06-10

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

Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

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

Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil

Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion proteinmore » for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.« less

Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

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

Browne, E.S.; Bhalla, V.K.

1991-02-01

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylatedmore » hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.« less

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

PubMed Central

Edwards, W. Barry

2013-01-01

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. PMID:23935860

Divergence between human and murine peroxisome proliferator-activated receptor alpha ligand specificities[S

PubMed Central

Oswal, Dhawal P.; Balanarasimha, Madhumitha; Loyer, Jeannette K.; Bedi, Shimpi; Soman, Frances L.; Rider, S. Dean; Hostetler, Heather A.

2013-01-01

Peroxisome proliferator-activated receptor α (PPARα) belongs to the family of ligand-dependent nuclear transcription factors that regulate energy metabolism. Although there exists remarkable overlap in the activities of PPARα across species, studies utilizing exogenous PPARα ligands suggest species differences in binding, activation, and physiological effects. While unsaturated long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) function as ligands for recombinant mouse PPARα (mPPARα), no such studies have been conducted with full-length human PPARα (hPPARα). The objective of the current study was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for hPPARα or whether there exist species differences for ligand specificity and affinity. Both hPPARα and mPPARα bound with high affinity to LCFA-CoA; however, differences were noted in LCFA affinities. A fluorescent LCFA analog was bound strongly only by mPPARα, and naturally occurring saturated LCFA was bound more strongly by hPPARα than mPPARα. Similarly, unsaturated LCFA induced transactivation of both hPPARα and mPPARα, whereas saturated LCFA induced transactivation only in hPPARα-expressing cells. These data identified LCFA and LCFA-CoA as endogenous ligands of hPPARα, demonstrated species differences in binding specificity and activity, and may help delineate the role of PPARα as a nutrient sensor in metabolic regulation. PMID:23797899

Improving binding mode and binding affinity predictions of docking by ligand-based search of protein conformations: evaluation in D3R grand challenge 2015

NASA Astrophysics Data System (ADS)

Xu, Xianjin; Yan, Chengfei; Zou, Xiaoqin

2017-08-01

The growing number of protein-ligand complex structures, particularly the structures of proteins co-bound with different ligands, in the Protein Data Bank helps us tackle two major challenges in molecular docking studies: the protein flexibility and the scoring function. Here, we introduced a systematic strategy by using the information embedded in the known protein-ligand complex structures to improve both binding mode and binding affinity predictions. Specifically, a ligand similarity calculation method was employed to search a receptor structure with a bound ligand sharing high similarity with the query ligand for the docking use. The strategy was applied to the two datasets (HSP90 and MAP4K4) in recent D3R Grand Challenge 2015. In addition, for the HSP90 dataset, a system-specific scoring function (ITScore2_hsp90) was generated by recalibrating our statistical potential-based scoring function (ITScore2) using the known protein-ligand complex structures and the statistical mechanics-based iterative method. For the HSP90 dataset, better performances were achieved for both binding mode and binding affinity predictions comparing with the original ITScore2 and with ensemble docking. For the MAP4K4 dataset, although there were only eight known protein-ligand complex structures, our docking strategy achieved a comparable performance with ensemble docking. Our method for receptor conformational selection and iterative method for the development of system-specific statistical potential-based scoring functions can be easily applied to other protein targets that have a number of protein-ligand complex structures available to improve predictions on binding.

Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.

PubMed

Raffaï, R; Weisgraber, K H; MacKenzie, R; Rupp, B; Rassart, E; Hirama, T; Innerarity, T L; Milne, R

2000-03-10

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.

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.

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.

Cell-specific targeting by heterobivalent ligands.

PubMed

Josan, Jatinder S; Handl, Heather L; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M; Vagner, Josef; Mash, Eugene A; Hruby, Victor J; Gillies, Robert J

2011-07-20

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Cell-Specific Targeting by Heterobivalent Ligands

PubMed Central

Josan, Jatinder S.; Handl, Heather L.; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M.; Vagner, Josef; Mash, Eugene A.; Hruby, Victor J.; Gillies, Robert J.

2012-01-01

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach—to specifically target combinations of cell-surface receptors using heteromultivalent ligands (“receptor combination approach”). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle4, DPhe7]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20–50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH2. Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging. PMID:21639139

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

The Universal Statistical Distributions of the Affinity, Equilibrium Constants, Kinetics and Specificity in Biomolecular Recognition

PubMed Central

Zheng, Xiliang; Wang, Jin

2015-01-01

We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity), the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics. PMID:25885453

Selection, Identification, and Binding Mechanism Studies of an ssDNA Aptamer Targeted to Different Stages of E. coli O157:H7.

PubMed

Zou, Ying; Duan, Nuo; Wu, Shijia; Shen, Mofei; Wang, Zhouping

2018-06-06

Enterohemorrhagic Escherichia coli O157:H7 ( E. coli O157:H7) is known as an important food-borne pathogen related to public health. In this study, aptamers which could bind to different stages of E. coli O157:H7 (adjustment phase, log phase, and stationary phase) with high affinity and specificity were obtained by the whole cell-SELEX method through 14 selection rounds including three counter-selection rounds. Altogether, 32 sequences were obtained, and nine families were classified to select the optimal aptamer. To analyze affinity and specificity by flow cytometer, an ssDNA aptamer named Apt-5 was picked out as the optimal aptamer that recognizes different stages of E. coli O157:H7 specifically with the K d value of 9.04 ± 2.80 nM. In addition, in order to study the binding mechanism, target bacteria were treated by proteinase K and trypsin, indicating that the specific binding site is not protein on the cell membrane. Furthermore, when we treated E. coli O157:H7 with EDTA, the result showed that the binding site might be lipopolysaccharide (LPS) on the outer membrane of E. coli O157:H7.

Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel.

PubMed

Henry, Pauline C; Kanelis, Voula; O'Brien, M Christine; Kim, Brian; Gautschi, Ivan; Forman-Kay, Julie; Schild, Laurent; Rotin, Daniela

2003-05-30

The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression by Nedd4 proteins.

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

Development and characterization of a camelid single-domain antibody directed to human CD22 biomarker.

PubMed

Faraji, Fatemeh; Tajik, Nader; Behdani, Mahdi; Shokrgozar, Mohammad Ali; Zarnani, Amir Hassan; Shahhosseini, Fatemeh; Habibi-Anbouhi, Mahdi

2018-03-15

CD22 is a B-cell-specific trans-membrane glycoprotein, which is found on the surface of the most B cells and modulates their function, survival, and apoptosis. Recently, targeting this cell surface biomarker in B-cell malignancies and disorders has attracted a lot of attention. The variable domain of camelid single-chain antibodies (VHH, nanobody) is a form of antibodies with novel properties including small size (15-17 kDa), thermal and chemical stability, high affinity and homology to human antibody sequences. In this study, a novel anti-CD22-specific VHH (Nb) has been developed and characterized by the screening of an immunized phage display library and its binding to CD22 + B cells is evaluated. Produced anti-CD22 VHH had a single protein band about 17 kDa of molecular size in Western blotting and its binding affinity was approximately 9 × 10 -9  M. Also, this product had high specificity and it was able to recognize the natural CD22 antigen in CD22+ cell lysate as well as on the cell surface (93%). This anti-CD22 VHH with both high affinity and specificity recognizes CD22 antigen well and can be used in diagnosis and treatment of B cell disorders and malignancies. © 2018 International Union of Biochemistry and Molecular Biology, Inc.

Nuclease-resistant c-di-AMP derivatives that differentially recognize RNA and protein receptors

PubMed Central

Meehan, Robert E.; Torgerson, Chad D.; Gaffney, Barbara L.; Jones, Roger A.; Strobel, Scott A.

2016-01-01

The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3’-5’-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activate specific pathways and mediate phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogs, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position, more than a carbonyl at the C6 position. We also identified phosphate-modified analogs that bind both the ydaO RNA and GdpP protein with high affinity, while symmetrically-modified ribose analogs exhibited a substantial decrease in ydaO affinity, but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogs could be useful as chemical tools to specifically target subsections of the second-messenger signaling pathways. PMID:26789423

Evaluation of galectin binding by frontal affinity chromatography (FAC).

PubMed

Iwaki, Jun; Hirabayashi, Jun

2015-01-01

Frontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail. As a result, their minimal requirements for recognition of disaccharides, i.e., β-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K d's for interactions between a series of standard glycans and various galectins.

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

PubMed

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

2004-06-01

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

Angiotensin II receptors in testes

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

Millan, M.A.; Aguilera, G.

Receptors for angiotensin II (AII) were identified and characterized in testes of rats and several primate species. Autoradiographic analysis of the binding of 125I-labeled (Sar1,Ile8)AII to rat, rhesus monkey, cebus monkey, and human testicular slide-mounted frozen sections indicated specific binding to Leydig cells in the interstitium. In rat collagenase-dispersed interstitial cells fractionated by Percoll gradient, AII receptor content was parallel to that of hCG receptors, confirming that the AII receptors are in the Leydig cells. In rat dispersed Leydig cells, binding was specific for AII and its analogs and of high affinity (Kd, 4.8 nM), with a receptor concentration ofmore » 15 fmol/10(6) cells. Studies of AII receptors in rat testes during development reveals the presence of high receptor density in newborn rats which decreases toward the adult age (4934 +/- 309, 1460 +/- 228, 772 +/- 169, and 82 +/- 12 fmol/mg protein at 5, 15, 20, and 30 days of age, respectively) with no change in affinity. At all ages receptors were located in the interstitium, and the decrease in binding was parallel to the decrease in the interstitial to tubular ratio observed with age. AII receptor properties in membrane-rich fractions from prepuberal testes were similar in the rat and rhesus monkey. Binding was time and temperature dependent, reaching a plateau at 60 min at 37 C, and was increased by divalent cations, EGTA, and dithiothreitol up to 0.5 mM. In membranes from prepuberal monkey testes, AII receptors were specific for AII analogs and of high affinity (Kd, 4.2 nM) with a receptor concentration of 7599 +/- 1342 fmol/mg protein. The presence of AII receptors in Leydig cells in rat and primate testes in conjunction with reports of the presence of other components of the renin-angiotensin system in the testes suggests that the peptide has a physiological role in testicular function.« less

Effect of Scoparia dulcis extract on insulin receptors in streptozotocin induced diabetic rats: studies on insulin binding to erythrocytes.

PubMed

Pari, Leelavinothan; Latha, Muniappan; Rao, Chippada Appa

2004-01-01

We investigated the insulin-receptor-binding effect of Scoparia dulcis plant extract in streptozotocin (STZ)-induced male Wistar rats, using circulating erythrocytes (ER) as a model system. An aqueous extract of S dulcis plant (SPEt) (200 mg/kg body weight) was administered orally. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors. Glibenclamide was used as standard reference drug. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (55.0 +/- 2.8%) than in SPEt-treated (70.0 +/- 3.5%)- and glibenclamide-treated (65.0 +/- 3.3%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with SPEt- and glibenclamide-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from SPEt and glibenclamide treated diabetic rats having 2.5 +/- 0.15 x 10(10) M(-1) (Kd1); 17.0 +/- 1.0 x 10(-8) M(-1) (Kd2), and 2.0 +/- 0.1 x 10(-10) M(-1) (Kd1); 12.3 +/- 0.9 x 10(-8) M(-1) (Kd2) compared with 1.0 +/- 0.08 x 10(-10) M(-1) (Kd1); 2.7 +/- 0.25 x 10(-8) M(-1) (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in STZ-induced diabetic rats. Treatment with SPEt and glibenclamide significantly improved specific insulin binding, with receptor number and affinity binding (p < 0.001) reaching almost normal non-diabetic levels. The data presented here show that SPEt and glibenclamide increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low Affinity Memory CD8+ T Cells During Heterologous Immunity

PubMed Central

Krummey, Scott M.; Chen, Ching-Wen; Guasch, Sara A.; Liu, Danya; Wagener, Maylene; Larsen, Christian P; Ford, Mandy L.

2016-01-01

The affinity of a T cell receptor (TCR) binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic antigen and mediate graft rejection. We found that low affinity primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared to high affinity primed memory T cells. Low affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low affinity primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low but not high affinity primed animals. These data establish a functional connection between TNF signaling and TCR priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation. PMID:27481849

High-Speed Lateral Flow Strategy for a Fast Biosensing with an Improved Selectivity and Binding Affinity.

PubMed

Cho, Dong Guk; Yoo, Haneul; Lee, Haein; Choi, Yeol Kyo; Lee, Minju; Ahn, Dong June; Hong, Seunghun

2018-05-10

We report a high-speed lateral flow strategy for a fast biosensing with an improved selectivity and binding affinity even under harsh conditions. In this strategy, biosensors were fixed at a location away from the center of a round shape disk, and the disk was rotated to create the lateral flow of a target solution on the biosensors during the sensing measurements. Experimental results using the strategy showed high reaction speeds, high binding affinity, and low nonspecific adsorptions of target molecules to biosensors. Furthermore, binding affinity between target molecules and sensing molecules was enhanced even in harsh conditions such as low pH and low ionic strength conditions. These results show that the strategy can improve the performance of conventional biosensors by generating high-speed lateral flows on a biosensor surface. Therefore, our strategy can be utilized as a simple but powerful tool for versatile bio and medical applications.

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.

Relationship between helix stability and binding affinities: molecular dynamics simulations of Bfl-1/A1-binding pro-apoptotic BH3 peptide helices in explicit solvent.

PubMed

Modi, Vivek; Lama, Dilraj; Sankararamakrishnan, Ramasubbu

2013-01-01

The anti-apoptotic protein Bfl-1, also known as A1, belongs to the Bcl-2 family of proteins and interacts with pro-apoptotic Bcl-2 counterparts to regulate programmed cell death. As demonstrated for other anti-apoptotic Bcl-2 proteins, Bfl-1/A1 has also been shown to be overexpressed in various human cancers and hence they are attractive targets for anticancer drugs. Peptides derived from the BH3 region of pro-apoptotic Bcl-2 proteins have been shown to elicit similar biological response as that of parent proteins. BH3 peptides from different pro-apoptotic proteins have wide range of affinities for Bfl-1/A1. Experimentally determined complex structures show that the hydrophobic side of amphipathic BH3 peptides binds to the hydrophobic groove formed by the α-helical bundle of Bfl-1/A1 protein. Apart from the length and amino acid composition, a BH3 peptide's ability to form a stable helical structure has been suggested to be important for its high binding affinity. Molecular dynamics simulations of three BH3 peptides derived from the pro-apoptotic proteins Bak, Bid, and Bmf were carried out each for a period of at least 100 ns after 2 ns equilibration run. The length of simulated BH3 peptides varied from 22 to 24 residues and their binding affinities for Bfl-1/A1 varied from 1 to 180 nM. Our results show that the hydrophobic residues from the hydrophobic face of BH3 peptides tend to cluster together quickly to avoid being exposed to the solvent. This resulted in either reduction of helix length or complete loss of helical character. Bak and Bid BH3 peptides with high affinities for Bf1-1/A1 have stable helical segments in the N-terminal region. The highly conserved Leu residue lies just outside the helical region at the C-terminal end. Capping interactions arising out of N-cap residues seem to be extremely important to maintain the helical stability. Favorable hydrophilic interactions between residues also give further stability to the helix fragment and at least one of the interacting residues resides within the helical region. Bmf BH3 peptide with a weaker binding affinity for Bmf-1/A1 completely lost its helical character at the end of 100 ns production run and a further 50 ns simulation showed that the Bmf peptide continues to remain in random conformation. The present study clearly establishes a link between a BH3 peptide's ability to form a stable helical segment and its high binding affinity for an anti-apoptotic protein. To further test this hypothesis, we simulated a mutant Bmf peptide for 100 ns in which two residues R129 and H146 were substituted by Asn in silico in the wild-type peptide. Introduction of N-terminal Asn clearly enabled the formation of capping interactions at the N-terminus and resulted in a stable N-terminal helical segment. This demonstrates that the knowledge of interactions that help to maintain stable helical segments in a high-affinity BH3 peptide will help in designing highly specific peptide-based drugs/inhibitors. Such molecules will have the ability to bind a particular anti-apoptotic protein with high affinity.

Isolation of an Aptamer that Binds Specifically to E. coli

PubMed Central

Cleto, Fernanda; Krieger, Marco Aurélio; Cardoso, Josiane

2016-01-01

Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers–called P12-31—is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies. PMID:27104834

Identification of STAT1 and STAT3 Specific Inhibitors Using Comparative Virtual Screening and Docking Validation

PubMed Central

Szelag, Malgorzata; Czerwoniec, Anna; Wesoly, Joanna; Bluyssen, Hans A. R.

2015-01-01

Signal transducers and activators of transcription (STATs) facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr)-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (h)STATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the ‘STAT-comparative binding affinity value’ and ‘ligand binding pose variation’ as selection criteria. In silico screening of a multi-million clean leads (CL) compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our understanding of the functional role of these STATs in different diseases and benefit the clinical need for more drugable STAT inhibitors with high specificity, potency and excellent bioavailability. PMID:25710482

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

PubMed Central

Kumar, Hemant; Finer-Moore, Janet S.; Kaback, H. Ronald; Stroud, Robert M.

2015-01-01

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent. PMID:26157133

Biomining of MoS2 with Peptide-based Smart Biomaterials.

PubMed

Cetinel, Sibel; Shen, Wei-Zheng; Aminpour, Maral; Bhomkar, Prasanna; Wang, Feng; Borujeny, Elham Rafie; Sharma, Kumakshi; Nayebi, Niloofar; Montemagno, Carlo

2018-02-20

Biomining of valuable metals using a target specific approach promises increased purification yields and decreased cost. Target specificity can be implemented with proteins/peptides, the biological molecules, responsible from various structural and functional pathways in living organisms by virtue of their specific recognition abilities towards both organic and inorganic materials. Phage display libraries are used to identify peptide biomolecules capable of specifically recognizing and binding organic/inorganic materials of interest with high affinities. Using combinatorial approaches, these molecular recognition elements can be converted into smart hybrid biomaterials and harnessed for biotechnological applications. Herein, we used a commercially available phage-display library to identify peptides with specific binding affinity to molybdenite (MoS 2 ) and used them to decorate magnetic NPs. These peptide-coupled NPs could capture MoS 2 under a variety of environmental conditions. The same batch of NPs could be re-used multiple times to harvest MoS 2 , clearly suggesting that this hybrid material was robust and recyclable. The advantages of this smart hybrid biomaterial with respect to its MoS 2 -binding specificity, robust performance under environmentally challenging conditions and its recyclability suggests its potential application in harvesting MoS 2 from tailing ponds and downstream mining processes.

Binding specificity of the juvenile hormone carrier protein from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae).

PubMed

Peterson, R C; Reich, M F; Dunn, P E; Law, J H; Katzenellnbogen, J A

1977-05-17

A series of analogues of insect juvenile hormone (four geometric isomers of methyl epoxyfarnesenate, several para-substituted epoxygeranyl phenyl ethers, and epoxyfarnesol and its acetate and haloacetate derivatives) was prepared to investigate the binding specificity of the hemolymph juvenile hormone binding protein from the tobacco hornworm Manduct sexta. The relative binding affinities were determined by a competition assay against radiolabeled methyl (E,E)-3,11-dimethyl-7-ethyl-cis-10,11-epoxytrideca-2,6-dienoate (JH I). The ratio of dissociation constants was estimated by plotting competitor data according to a linear transformation of the dissociation equations describing competition of two ligands for a binding protein. The importance of the geometry of the sesquiterpene hydrocarbon chain is indicated by the fact that the binding affinity is decreased as Z (cis) double bonds are substituted for E (trans) double bonds in the methyl epoxyfarnesenate series; the unepoxidized analogues do not bind. A carboxylic ester function is important although its orientation can be reversed, as indicated by the good binding of epoxyfarnesyl acetate. In the monoterpene series, methyl epoxygeranoate shows no affinity for the binding protein, but substitution of a phenyl or p-carbomethoxyphenyl ether for the ester function imparts a low, but significant affinity. These data taken together with earlier results indicate that the binding site for juvenile hormone in the hemolymph binding protein is characterized by a sterically defined hydrophobic region with polar sites that recognize the epoxide and the ester functions.

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action.

PubMed

Pai, Sudipta; Das, Mili; Banerjee, Rahul; Dasgupta, Dipak

2011-08-01

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.

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

Binding of calcium and target peptide to calmodulin-like protein CML19, the centrin 2 of Arabidopsis thaliana.

PubMed

La Verde, Valentina; Trande, Matteo; D'Onofrio, Mariapina; Dominici, Paola; Astegno, Alessandra

2018-03-01

Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein, the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level, little is known at a molecular level. Herein, we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca 2+ -specific binding sites, two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca 2+ to CML19 increases its alpha-helix content, stabilizes the tertiary structure, and triggers a conformational change, resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4, and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca 2+ (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19. Copyright © 2017 Elsevier B.V. All rights reserved.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2008-02-12

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2012-01-31

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

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.

Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

PubMed

Ciossek, T; Ullrich, A

1997-01-09

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein.

PubMed

Toscano-Garibay, Julia D; Benítez-Hess, María L; Alvarez-Salas, Luis M

2011-02-01

Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer. Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.

Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

PubMed Central

Gao, Rong

2015-01-01

ABSTRACT Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. PMID:26015501

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

PubMed

Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

2018-01-01

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

Structural and functional evidences for the interactions between nuclear hormone receptors and endocrine disruptors at low doses.

PubMed

Balaguer, Patrick; Delfosse, Vanessa; Grimaldi, Marina; Bourguet, William

Endocrine-disrupting chemicals (EDCs) represent a broad class of exogenous substances that cause adverse effects in the endocrine system mainly by interacting with nuclear hormone receptors (NRs). Humans are generally exposed to low doses of pollutants, and current researches aim at deciphering the mechanisms accounting for the health impact of EDCs at environmental concentrations. Our correlative analysis of structural, interaction and cell-based data has revealed a variety of, sometimes unexpected, binding modes, reflecting a wide range of EDC affinities and specificities. Here, we present a few representative examples to illustrate various means by which EDCs achieve high-affinity binding to NRs. These examples include the binding of the mycoestrogen α-zearalanol to estrogen receptors, the covalent interaction of organotins with the retinoid X- and peroxisome proliferator-activated receptors, and the cooperative binding of two chemicals to the pregnane X receptor. We also discuss some hypotheses that could further explain low-concentration effects of EDCs with weaker affinity towards NRs. Copyright © 2017. Published by Elsevier Masson SAS.

Identification of a receptor for ADP on blood platelets by photoaffinity labelling.

PubMed Central

Cristalli, G; Mills, D C

1993-01-01

The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase. Images Figure 5 PMID:8387782

Insights from the predicted structural analysis of carborane substituted withaferin A with Indoleamine - 2,3-dioxygenase as a potent inhibitor.

PubMed

Basha, Syed Hussain; Thakur, Abhishek; Samad, Firoz A

2016-01-01

Indoleamine-2,3-dioxygenase (IDO) an immunoregulatory enzyme and emerging as a new therapeutic drug target for the treatment of cancer. Carboranes, an icosahedral arrangement of eleven boron atoms plus one carbon atom with unique pharmacological properties such low toxicity, isosterism with phenyl ring and stability to hydrolysis. On the other hand, carboranes are known to increase the interaction of ligand with non-polar region of the protein provides an excellent platform to explore these carboranes towards designing and development of novel, potent and target specific drug candidates with further enhanced binding affinities. Despite of their many potential applications, molecular modeling studies of carborane-substituted ligands with macromolecules have been rarely reported. Previously, we have demonstrated the promising high binding affinity of Withaferin-A (WA) for IDO. In this present study, we investigated the effect of carborane substitutions on WA compound towards developing novel analogs for target specific IDO inhibition with better potency. Interesting docked poses and molecular interactions for the carborane substituted WA ligands were elucidated. Based on our In-silico studies, carborane substituted at various position of WA has shown enhanced binding affinity towards IDO, worth of considering for further studies.

Insights from the predicted structural analysis of carborane substituted withaferin A with Indoleamine - 2,3-dioxygenase as a potent inhibitor

PubMed Central

Samad, Firoz A

2016-01-01

Indoleamine-2,3-dioxygenase (IDO) an immunoregulatory enzyme and emerging as a new therapeutic drug target for the treatment of cancer. Carboranes, an icosahedral arrangement of eleven boron atoms plus one carbon atom with unique pharmacological properties such low toxicity, isosterism with phenyl ring and stability to hydrolysis. On the other hand, carboranes are known to increase the interaction of ligand with non-polar region of the protein provides an excellent platform to explore these carboranes towards designing and development of novel, potent and target specific drug candidates with further enhanced binding affinities. Despite of their many potential applications, molecular modeling studies of carborane-substituted ligands with macromolecules have been rarely reported. Previously, we have demonstrated the promising high binding affinity of Withaferin-A (WA) for IDO. In this present study, we investigated the effect of carborane substitutions on WA compound towards developing novel analogs for target specific IDO inhibition with better potency. Interesting docked poses and molecular interactions for the carborane substituted WA ligands were elucidated. Based on our In-silico studies, carborane substituted at various position of WA has shown enhanced binding affinity towards IDO, worth of considering for further studies. PMID:28250615

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.

PubMed

Ahmad, Kareem M; Xiao, Yi; Soh, H Tom

2012-12-01

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K(d)) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

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

Characterization of glucagon-like peptide-1 receptor-binding determinants.

PubMed

Xiao, Q; Jeng, W; Wheeler, M B

2000-12-01

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.

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

Circular permutation of the starch-binding domain: inversion of ligand selectivity with increased affinity.

PubMed

Stephen, Preyesh; Tseng, Kai-Li; Liu, Yu-Nan; Lyu, Ping-Chiang

2012-03-07

Proteins containing starch-binding domains (SBDs) are used in a variety of scientific and technological applications. A circularly permutated SBD (CP90) with improved affinity and selectivity toward longer-chain carbohydrates was synthesized, suggesting that a new starch-binding protein may be developed for specific scientific and industrial applications. This journal is © The Royal Society of Chemistry 2012

Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

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

Bach, Christian; Sherman, William; Pallis, Jani

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable toolsmore » to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.« less

Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

DOE PAGES

Bach, Christian; Sherman, William; Pallis, Jani; ...

2014-01-01

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable toolsmore » to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.« less

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

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

Mullinax, R.L.; Gross, E.A.; Amberg, J.R.

1990-10-01

The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These humanmore » antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.« less

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

Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions

PubMed Central

Joseph, Prem Raj B.; Mosier, Philip D.; Desai, Umesh R.; Rajarathnam, Krishna

2015-01-01

Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8–GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer–GAG interactions and function. PMID:26371375

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.

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

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

Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

1990-02-20

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

A specific l-tri-iodothyronine-binding protein in the cytosol fraction of human breast adipose tissue

PubMed Central

Rao, Marie Luise; Rao, Govind S.

1982-01-01

1. Binding of l-tri-[125I]iodothyronine to the cytosol fraction of normal human female breast adipose tissue was investigated by the charcoal adsorption method. Equilibrium of binding was reached after 120s at 25°C. 2. The l-tri-[125I]iodothyronine-binding component is a protein; this was confirmed by experiments in which binding was totally lost after heating the cytosol fraction for 10min at 100°C and in which binding was diminished after treatment with proteolytic enzymes and with thiol-group-blocking reagents. The binding protein was stable at −38°C for several months. 3. It displayed saturability, high affinity (apparent Kd 3.28nm) and a single class of binding sites. 4. High specificity for l-tri-iodothyronine and l-3,5-di-iodo-3′-isopropylthyronine was observed, whereas other iodothyronines were less effective in displacing l-tri-[125I]-iodothyronine from its binding site. 5. The binding of the hormone by the cytosol fraction did not show a pH optimum. 6. When cytosol fractions of adipose tissue from different females were subjected to radioimmunoassay for the determination of thyroxine-binding globulin a value of 0.304±0.11μg/mg of cytosol protein (mean±s.d., n=4) was obtained; the mean concentration in plasma was 0.309±0.07μg/mg of plasma protein (mean±s.d., n=3). 7. The Ka value of 6.3×108m−1 of l-tri-[125I]iodothyronine for binding to plasma, the similar thermalinactivation profiles of binding and the reactivity to thiol-group-blocking reagents were some properties common between the binding components from the cytosol fraction and plasma. 8. These results suggest that the cytosol fraction of human female breast adipose tissue contains thyroxine-binding globulin; the protein that binds l-tri-[125I]iodothyronine with high affinity and specificity appears to be similar to thyroxine-binding globulin. PMID:6289813

Identification of two H3-histamine receptor subtypes

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

West, R.E. Jr.; Zweig, A.; Shih, N.Y.

The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-(3H)methylhistamine ((3H)NAMHA). The results of (3H)NAMHA saturation binding and NAMHA inhibition of (3H)NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of (3H)NAMHA binding by the specific high affinity H3 antagonist thioperamide revealedmore » two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for (3H) NAMHA was reduced less than 2-fold, whereas (3H)NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data.« less

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

PubMed Central

VanScyoc, Wendy S; Sorensen, Brenda R; Rusinova, Elena; Laws, William R; Ross, J B Alexander; Shea, Madeline A

2002-01-01

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition. PMID:12414709

Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein

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

Bates, John T.; Keefer, Christopher J.; Slaughter, James C.

2014-04-15

The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (K{sub on}) for binding to RSV F protein, while alteration of dissociation rate (K{sub off}) did not significantly affect neutralizing activity. Interestingly, linkage of reduced K{sub on}more » with reduced potency mirrored the effect of increased K{sub on} found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants. - Highlights: • The relationship of affinity to neutralization for virus antibodies is uncertain. • Palivizumab binds to RSV escape mutant fusion proteins, but with reduced affinity. • Association rate (K{sub on}) correlated well with the potency of neutralization.« less

Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

PubMed

Gadermaier, E; Marth, K; Lupinek, C; Campana, R; Hofer, G; Blatt, K; Smiljkovic, D; Roder, U; Focke-Tejkl, M; Vrtala, S; Keller, W; Valent, P; Valenta, R; Flicker, S

2018-01-09

Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients. © 2018 The Authors. Allergy Published by John Wiley & Sons Ltd.

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.

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

Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity

PubMed Central

Kalra, Priya; Dhiman, Abhijeet; Cho, William C.; Bruno, John G.; Sharma, Tarun K.

2018-01-01

Aptamers are structured nucleic acid molecules that can bind to their targets with high affinity and specificity. However, conventional SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods may not necessarily produce aptamers of desired affinity and specificity. Thus, to address these questions, this perspective is intended to suggest some approaches and tips along with novel selection methods to enhance evolution of aptamers. This perspective covers latest novel innovations as well as a broad range of well-established approaches to improve the individual binding parameters (aptamer affinity, avidity, specificity and/or selectivity) of aptamers during and/or post-SELEX. The advantages and limitations of individual aptamer selection methods and post-SELEX optimizations, along with rational approaches to overcome these limitations are elucidated in each case. Further the impact of chosen selection milieus, linker-systems, aptamer cocktails and detection modules utilized in conjunction with target-specific aptamers, on the overall assay performance are discussed in detail, each with its own advantages and limitations. The simple variations suggested are easily available for facile implementation during and/or post-SELEX to develop ultrasensitive and specific assays. Finally, success studies of established aptamer-based assays are discussed, highlighting how they utilized some of the suggested methodologies to develop commercially successful point-of-care diagnostic assays. PMID:29868605

Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity.

PubMed

Kalra, Priya; Dhiman, Abhijeet; Cho, William C; Bruno, John G; Sharma, Tarun K

2018-01-01

Aptamers are structured nucleic acid molecules that can bind to their targets with high affinity and specificity. However, conventional SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods may not necessarily produce aptamers of desired affinity and specificity. Thus, to address these questions, this perspective is intended to suggest some approaches and tips along with novel selection methods to enhance evolution of aptamers. This perspective covers latest novel innovations as well as a broad range of well-established approaches to improve the individual binding parameters (aptamer affinity, avidity, specificity and/or selectivity) of aptamers during and/or post-SELEX. The advantages and limitations of individual aptamer selection methods and post-SELEX optimizations, along with rational approaches to overcome these limitations are elucidated in each case. Further the impact of chosen selection milieus, linker-systems, aptamer cocktails and detection modules utilized in conjunction with target-specific aptamers, on the overall assay performance are discussed in detail, each with its own advantages and limitations. The simple variations suggested are easily available for facile implementation during and/or post-SELEX to develop ultrasensitive and specific assays. Finally, success studies of established aptamer-based assays are discussed, highlighting how they utilized some of the suggested methodologies to develop commercially successful point-of-care diagnostic assays.

Quantifying Intrinsic Specificity: A Potential Complement to Affinity in Drug Screening

NASA Astrophysics Data System (ADS)

Wang, Jin; Zheng, Xiliang; Yang, Yongliang; Drueckhammer, Dale; Yang, Wei; Verkhivker, Gennardy; Wang, Erkang

2007-11-01

We report here the investigation of a novel description of specificity in protein-ligand binding based on energy landscape theory. We define a new term, intrinsic specificity ratio (ISR), which describes the level of discrimination in binding free energies of the native basin for a protein-ligand complex from the weaker binding states of the same ligand. We discuss the relationship between the intrinsic specificity we defined here and the conventional definition of specificity. In a docking study of molecules with the enzyme COX-2, we demonstrate a statistical correspondence between ISR value and geometrical shapes of the small molecules binding to COX-2. We further observe that the known selective (nonselective) inhibitors of COX-2 have higher (lower) ISR values. We suggest that intrinsic specificity ratio may be a useful new criterion and a complement to affinity in drug screening and in searching for potential drug lead compounds.

Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

PubMed

Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

2015-07-07

Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets.

PubMed

Nelson, Christopher S; Fuller, Chris K; Fordyce, Polly M; Greninger, Alexander L; Li, Hao; DeRisi, Joseph L

2013-07-01

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein's DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2's-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved.

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets

PubMed Central

Nelson, Christopher S.; Fuller, Chris K.; Fordyce, Polly M.; Greninger, Alexander L.; Li, Hao; DeRisi, Joseph L.

2013-01-01

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein’s DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2’s-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved. PMID:23625967

Toxic metals (Ni2+, Pb2+, Hg2+) binding affinity of dissolved organic matter (DOM) derived from different ages municipal landfill leachate

NASA Astrophysics Data System (ADS)

Rikta, S. Y.; Tareq, Shafi M.; Uddin, M. Khabir

2018-03-01

Solid waste production is rapidly increasing in Bangladesh and landfill leachate is the consequence of the decomposition of this waste. These leachates contain heavy metals and significant amount of dissolved organic matter (DOM). DOM is known to have considerable role in heavy metals speciation. Hence, it is important to characterize DOM/leachate and evaluate toxic metals binding affinity of DOM. The objectives of this study were to characterize the DOM in landfill leachate through physico-chemical and optical analyses and to investigate the toxic metals (Ni2+, Pb2+ and Hg2+) binding affinity of three different ages (fresh sample L-1, young sample L-2 and mature sample L-3) DOM samples. Results suggested that leachate is a potential pollutant which contained very high organic pollutant load. Conditional stability constant (Log K) and percentages of fluorophores that correspond to metal binding (% f) values indicated that young DOM sample (L-2) had the highest binding affinity to all the three metals ions. In general, DOM samples showed the following order affinity to the metal ions; Ni2+ binding affinity: L-2 > L-3 > L-1, Pb2+ binding affinity: L-2 > L-3 > L-1 and Hg2+ binding affinity: L-2 > L-1 > L-3.

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

PubMed Central

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

2005-01-01

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

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.

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

PubMed Central

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

2016-01-01

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

Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning.

PubMed

Koenig, Patrick; Sanowar, Sarah; Lee, Chingwei V; Fuh, Germaine

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with K D in the sub-nanomolar range. However, it also exhibits significant (K D of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.

Application of binding free energy calculations to prediction of binding modes and affinities of MDM2 and MDMX inhibitors.

PubMed

Lee, Hui Sun; Jo, Sunhwan; Lim, Hyun-Suk; Im, Wonpil

2012-07-23

Molecular docking is widely used to obtain binding modes and binding affinities of a molecule to a given target protein. Despite considerable efforts, however, prediction of both properties by docking remains challenging mainly due to protein's structural flexibility and inaccuracy of scoring functions. Here, an integrated approach has been developed to improve the accuracy of binding mode and affinity prediction and tested for small molecule MDM2 and MDMX antagonists. In this approach, initial candidate models selected from docking are subjected to equilibration MD simulations to further filter the models. Free energy perturbation molecular dynamics (FEP/MD) simulations are then applied to the filtered ligand models to enhance the ability in predicting the near-native ligand conformation. The calculated binding free energies for MDM2 complexes are overestimated compared to experimental measurements mainly due to the difficulties in sampling highly flexible apo-MDM2. Nonetheless, the FEP/MD binding free energy calculations are more promising for discriminating binders from nonbinders than docking scores. In particular, the comparison between the MDM2 and MDMX results suggests that apo-MDMX has lower flexibility than apo-MDM2. In addition, the FEP/MD calculations provide detailed information on the different energetic contributions to ligand binding, leading to a better understanding of the sensitivity and specificity of protein-ligand interactions.

Revealing multi-binding sites for taspine to VEGFR-2 by cell membrane chromatography zonal elution.

PubMed

Du, Hui; Wang, Sicen; Ren, Jing; Lv, Nan; He, Langchong

2012-03-01

A new high-expression vascular endothelial growth factor receptor-2 (VEGFR-2) cell membrane chromatography (CMC) method was developed to investigate the affinity of ligands for VEGFR-2. An HEK293 VEGFR-2/CMC system was applied to specifically recognize ligands acting on VEGFR-2. Sorafenib was used as a mobile phase additive to evaluate the effect of the marker's concentration on the retention of sorafenib and taspine, respectively. The relationship among the retention, the types of binding sites and the affinity of taspine binding to VEGFR-2 has also been concerned. The retention behavior indicated that sorafenib had two major binding regions on VEGFR-2, and that taspine might act as a multi-target VEGFR-2 inhibitor with similar biological activity to sorafenib. The equilibrium dissociation constants (K(D)) obtained from the model are (5.25 ± 0.31) × 10⁻⁷ and (9.88 ± 0.54) × 10⁻⁵ mol L⁻¹ for sorafenib at the high- and low-affinity sites, respectively, and the corresponding values for taspine are (3.88 ± 0.31) × 10⁻⁶ and (7.04 ± 0.49)×10⁻⁵ mol L⁻¹. The two types of binding sites contributed about a 1:2 ratio on the retention of taspine. Copyright © 2012 Elsevier B.V. All rights reserved.

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

PubMed Central

Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

2013-01-01

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response. PMID:23746516

Advances in Synthetic Peptides Reagent Discovery

DTIC Science & Technology

2013-07-01

to promote specific and high affinity binding. Longer incubations may result in nonspecific attachment, such as early biofilm formation. Because...peptide libraries yields ligand arrays that classify breast tumor subtypes,” Molecular Cancer Therapeutics, 8(5), 1312-1318 (2009). [26] J. M. Kogot

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

PubMed Central

Pintor, J.; Torres, M.; Castro, E.; Miras-Portugal, M. T.

1991-01-01

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site. PMID:1912985

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

DOE PAGES

Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.; ...

2016-02-09

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. Thismore » “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.« less

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

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

Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. Thismore » “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.« less

Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

NASA Astrophysics Data System (ADS)

Stefanescu, Raluca; Born, Rita; Moise, Adrian; Ernst, Beat; Przybylski, Michael

2011-01-01

Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

The sodium channel in membranes of electroplax. Binding of batrachotoxinin-a [(3)H]benzoate to particulate preparations from electric eel (electrophorus).

PubMed

McNeal, E T; Daly, J W

1986-01-01

Batrachotoxinin-A [(3)H]benzoate ([(3)H]BTX-B) binds specifically and with high affinity (K(D) 48 nM) to sites (B(max) 2.1 pmol/mg protein) associated with voltage-dependent sodium channels in rodent brain vesicular preparations. High affinity binding requires the presence of scorpion (Leiurus) venom and a membrane potential. Local anesthetics antagonize the binding. Nonspecific binding is defined in the presence of veratridine. In particulate preparations from electroplax of the eel Electrophorus electricus, [(3)H]BTX-B binds with a K(D) of about 140 nM and a B(max) of 2.5 pmol/mg protein in the presence of scorpion venom. Higher concentrations of scorpion venom are required to enhance binding in Electrophorus preparations than in brain preparations. Local anesthetics antagonize binding in Electrophorus preparations with potencies similar to those in brain preparations. Veratridine and batrachotoxin are less potent in blocking binding in Electrophorus than in brain preparations. It appears likely that binding in Electrophorus preparations is primarily to membrane fragments rather than vesicular entities as in brain. Binding of [(3)H]BTX-B to particulate preparations from electroplax of the ray Torpedo californica and the catfish Malapterurus electricus is mainly nonspecific. Scorpion venom does not enhance total binding and local anesthetics are not effective in antagonizing binding.

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

Fedynyshyn, J.P.

The opioid binding characteristics of the rat (PAG) and the signal transduction mechanisms of the opioid receptors were examined with in vitro radioligand binding, GTPase, adenylyl cyclase, and inositol phosphate assays. The nonselective ligand {sup 3}H-ethylketocyclazocine (EKC), the {mu} and {delta} selective ligand {sup 3}H-(D-Ala{sup 2}, D-Leu{sup 5}) enkephalin (DADLE), the {mu} selective ligand {sup 3}H-(D-Ala{sup 2}, N-methyl Phe{sup 4}, Glyol{sup 5}) enkephalin (DAGO), and the {delta} selective ligand {sup 3}H-(D-Pen{sup 2}, D-Pen{sup 5}) enkephalin (DPDPE) were separately used as tracer ligands to label opioid binding sites in rat PAG enriched P{sub 2} membrane in competition with unlabeled DADLE, DAGO,more » DPDPE, or the {kappa} selective ligand trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide, methane sulfonate, hydrate (U50, 488H). Only {mu} selective high affinity opioid binding was observed. No high affinity {delta} or {kappa} selective binding was detected. {sup 3}H-DAGO was used as a tracer ligand to label {mu} selective high affinity opioid binding sites in PAG enriched P{sub 2} membrane in competition with unlabeled {beta}-endorphin, dynorphin A (1-17), BAM-18, methionine enkephalin, dynorphin A (1-8), and leucine enkephalin. Of these endogenous opioid peptides only those with previously reported high affinity {mu} type opioid binding activity competed with {sup 3}H-DAGO for binding sites in rat PAG enriched P{sub 2} membrane with affinities similar to that of unlabeled DAGO.« less

Effects of salts on protein-surface interactions: applications for column chromatography.

PubMed

Tsumoto, Kouhei; Ejima, Daisuke; Senczuk, Anna M; Kita, Yoshiko; Arakawa, Tsutomu

2007-07-01

Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. Copyright 2007 Wiley-Liss, Inc.

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

PubMed

Lippe, Jan; Seichter, Wilhelm; Mazik, Monika

2015-12-28

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

Acquisition of Human-Type Receptor Binding Specificity by New H5N1 Influenza Virus Sublineages during Their Emergence in Birds in Egypt

PubMed Central

Watanabe, Yohei; Ibrahim, Madiha S.; Ellakany, Hany F.; Kawashita, Norihito; Mizuike, Rika; Hiramatsu, Hiroaki; Sriwilaijaroen, Nogluk; Takagi, Tatsuya; Suzuki, Yasuo; Ikuta, Kazuyoshi

2011-01-01

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential. PMID:21637809

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket.

PubMed

Soin, Thomas; Iga, Masatoshi; Swevers, Luc; Rougé, Pierre; Janssen, Colin R; Smagghe, Guy

2009-08-01

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.

Selection of peptidoglycan-specific aptamers for bacterial cells identification.

PubMed

Ferreira, Iêda Mendes; de Souza Lacerda, Camila Maria; de Faria, Lígia Santana; Corrêa, Cristiane Rodrigues; de Andrade, Antero Silva Ribeiro

2014-12-01

Peptidoglycan is a highly complex and essential macromolecule of bacterial outer cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by peptides. Peptidoglycan has a particular composition which makes it a possible target for specific bacterial recognition. Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Aptamers can be labeled with different radioisotopes and possess several properties that make them suitable for molecular imaging. The purpose of this study was to obtain aptamers for use as radiopharmaceutical in bacterial infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was 0.415 + 0.047 μM and for Antibac2 was 1.261 + 0.280 μM. These aptamers labeled with (32)P showed high affinity for Staphylococcus aureus cells. The binding to S. aureus and Escherichia coli in vitro were significantly higher than for Candida albicans and human fibroblasts, demonstrating their specificity for bacterial cells. These results point Antibac1 and Antibac2 as promising tools for bacterial infections identification.

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.

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).

Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

PubMed

Horejsí, V; Tichá, M; Kocourek, J

1977-09-29

Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

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

Branchek, T.; Adham, N.; Macchi, M.

1990-11-01

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.

PubMed

Takakusagi, Yoichi; Kuramochi, Kouji; Takagi, Manami; Kusayanagi, Tomoe; Manita, Daisuke; Ozawa, Hiroko; Iwakiri, Kanako; Takakusagi, Kaori; Miyano, Yuka; Nakazaki, Atsuo; Kobayashi, Susumu; Sugawara, Fumio; Sakaguchi, Kengo

2008-11-15

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.

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

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

PubMed

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

1988-07-01

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

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line.

PubMed

Kumar, Ashok; Bansal, Vibha; Andersson, Jonatan; Roychoudhury, Pradip K; Mattiasson, Bo

2006-01-20

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.

Two classes of binding sites for [3H]substance P in rat cerebral cortex.

PubMed

Geraghty, D P; Burcher, E

1993-01-22

The binding characteristics of [3H]substance P ([3H]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [3H]SP reached equilibrium after 50 min at 25 degrees C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, Kd, 0.22 nM) and low affinity sites (Kd, 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [3H]SP binding. The rank order of potency for inhibition of [3H]SP (2 nM) binding was SP > or = [Sar9, Met(O2)11]-SP > or = physalaemin > SP(3-11) > NP gamma = [Ala3]-SP > or = SP(4-11) > or = NPK > or = SP(5-11) > or = NKB approximately NKA > SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [3H]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9, Met(O2)11]-SP could be resolved into two components.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of phycoerythrin and its conjugates to murine low affinity receptors for immunoglobulin G.

PubMed

Takizawa, F; Kinet, J P; Adamczewski, M

1993-06-18

Conjugates of R-phycoerythrin are widely used for immunohistochemistry, especially for two-color flow cytometry. Their use is however limited by their apparent tendency to bind non-specifically. Using cells transfected with cDNAs for the murine low affinity receptors for immunoglobulin G (Fc gamma RII and -III) and cells naturally expressing these receptors, we demonstrate that R-phycoerythrin and its conjugates bind specifically and inhibitably to Fc gamma RII and -III. Immunofluorescence stainings of cells bearing these receptors, such as macrophages, monocytes, neutrophils, mast cells, subsets of T cells, and natural killer cells, may therefore not reflect the binding of antibody to antigen, but rather the binding of R-phycoerythrin to the receptors.

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe.

PubMed

Zilio, Nicola; Boddy, Michael N

2017-03-01

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins. © 2017 Cold Spring Harbor Laboratory Press.

Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as Fc gamma RII and Fc gamma RIII.

PubMed

Takizawa, F; Adamczewski, M; Kinet, J P

1992-08-01

In addition to their well characterized high affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) mast cells have long been suspected to express undefined Fc receptors capable of binding IgE with low affinity. In this paper, we show that Fc gamma RII and Fc gamma RIII, but not Mac-2, on mouse mast cells and macrophages bind IgE-immune complexes. This binding is efficiently competed by 2.4G2, a monoclonal antibody against the extracellular homologous region of both Fc gamma RII and Fc gamma RIII. Furthermore, IgE-immune complexes bind specifically to Fc gamma RII or Fc gamma RIII transfected into COS-7 cells. The association constants of IgE binding estimated from competition experiments are about 3.1 x 10(5) M-1 for Fc gamma RII, and 4.8 x 10(5) M-1 for Fc gamma RIII. Engagement of Fc gamma RII and Fc gamma RIII with IgE-immune complexes (after blocking access to Fc epsilon RI) or with IgG-immune complexes triggers C57.1 mouse mast cells to release serotonin. This release is inhibited by 2.4G2, and at maximum, reaches 30-40% of the intracellular content, about half of the maximal release (60-80%) obtained after Fc epsilon RI engagement. These data demonstrate that mouse Fc gamma RII and Fc gamma RIII are not isotype specific, and that the binding of IgE-immune complexes to these receptors induces cell activation.

Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as Fc gamma RII and Fc gamma RIII

PubMed Central

1992-01-01

In addition to their well characterized high affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) mast cells have long been suspected to express undefined Fc receptors capable of binding IgE with low affinity. In this paper, we show that Fc gamma RII and Fc gamma RIII, but not Mac-2, on mouse mast cells and macrophages bind IgE-immune complexes. This binding is efficiently competed by 2.4G2, a monoclonal antibody against the extracellular homologous region of both Fc gamma RII and Fc gamma RIII. Furthermore, IgE-immune complexes bind specifically to Fc gamma RII or Fc gamma RIII transfected into COS-7 cells. The association constants of IgE binding estimated from competition experiments are about 3.1 x 10(5) M-1 for Fc gamma RII, and 4.8 x 10(5) M-1 for Fc gamma RIII. Engagement of Fc gamma RII and Fc gamma RIII with IgE-immune complexes (after blocking access to Fc epsilon RI) or with IgG-immune complexes triggers C57.1 mouse mast cells to release serotonin. This release is inhibited by 2.4G2, and at maximum, reaches 30-40% of the intracellular content, about half of the maximal release (60-80%) obtained after Fc epsilon RI engagement. These data demonstrate that mouse Fc gamma RII and Fc gamma RIII are not isotype specific, and that the binding of IgE-immune complexes to these receptors induces cell activation. PMID:1386873

Biochemical study of prolactin binding sites in Xenopus laevis brain and choroid plexus

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

Muccioli, G.; Guardabassi, A.; Pattono, P.

1990-03-01

The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrencemore » of prolactin specific binding sites in Xenopus laevis choroid plexus cells.« less

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

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

PubMed

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

2017-09-20

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

Structure of adenovirus bound to cellular receptor car

DOEpatents

Freimuth, Paul I.

2007-01-02

Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

PubMed

Chang, C P; Hüsler, T; Zhao, J; Wiedmer, T; Sims, P J

1994-10-21

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

PubMed

Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

2016-01-01

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

Bovine IgG subclasses and fertility of Echinococcus granulosus hydatid cysts.

PubMed

Riesle, Silke; García, María Pía; Hidalgo, Christian; Galanti, Norbel; Saenz, Leonardo; Paredes, Rodolfo

2014-09-15

Hydatidosis is an important zoonotic disease of worldwide distribution, causing important health problems to humans and major economical losses in infected livestock. Echinococcus granulosus, the etiological agent of hydatid disease, induces a humoral immune response in the intermediate host (human and herbivorous) against hydatid cyst antigens. Specifically, IgGs are found in the laminar and germinal layers and inside the lumen of fertile and infertile hydatid cysts. In the germinal layer of infertile cysts IgGs are found in an order of magnitude greater than in the germinal layer of fertile cysts; a fraction of those IgGs are associated with high affinity to germinal layer proteins, suggesting their binding to specific parasite antigens. We have previously shown that those immunoglobulins, bound with high affinity to the germinal layer of hydatid cysts, induce apoptosis leading to cyst infertility. In the present work the presence of IgG1 and IgG2 subclasses in the germinal layer of both fertile and infertile hydatid cysts is reported. IgG1 is the most relevant immunoglobulin subclass present in the germinal layer of infertile cysts and bound with high affinity to that parasite structure. Contrarily, though the IgG2 subclass was also found in the germinal and adventitial layers, those immunoglobulins show low affinity to parasite antigens. We propose that the binding of an IgG1 subclass to parasite antigens present in the germinal layer is involved in the mechanism of cyst infertility. Copyright © 2014 Elsevier B.V. All rights reserved.

A comparative study of family-specific protein-ligand complex affinity prediction based on random forest approach

NASA Astrophysics Data System (ADS)

Wang, Yu; Guo, Yanzhi; Kuang, Qifan; Pu, Xuemei; Ji, Yue; Zhang, Zhihang; Li, Menglong

2015-04-01

The assessment of binding affinity between ligands and the target proteins plays an essential role in drug discovery and design process. As an alternative to widely used scoring approaches, machine learning methods have also been proposed for fast prediction of the binding affinity with promising results, but most of them were developed as all-purpose models despite of the specific functions of different protein families, since proteins from different function families always have different structures and physicochemical features. In this study, we proposed a random forest method to predict the protein-ligand binding affinity based on a comprehensive feature set covering protein sequence, binding pocket, ligand structure and intermolecular interaction. Feature processing and compression was respectively implemented for different protein family datasets, which indicates that different features contribute to different models, so individual representation for each protein family is necessary. Three family-specific models were constructed for three important protein target families of HIV-1 protease, trypsin and carbonic anhydrase respectively. As a comparison, two generic models including diverse protein families were also built. The evaluation results show that models on family-specific datasets have the superior performance to those on the generic datasets and the Pearson and Spearman correlation coefficients ( R p and Rs) on the test sets are 0.740, 0.874, 0.735 and 0.697, 0.853, 0.723 for HIV-1 protease, trypsin and carbonic anhydrase respectively. Comparisons with the other methods further demonstrate that individual representation and model construction for each protein family is a more reasonable way in predicting the affinity of one particular protein family.

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.

Binding site size limit of the 2:1 pyrrole-imidazole polyamide-DNA motif.

PubMed Central

Kelly, J J; Baird, E E; Dervan, P B

1996-01-01

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Six polyamides containing three to eight rings bind DNA sites 5-10 bp in length, respectively. Quantitative DNase I footprint titration experiments demonstrate that affinity maximizes and is similar at ring sizes of five, six, and seven. Sequence specificity decreases as the length of the polyamides increases beyond five rings. These results provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity. Images Fig. 4 PMID:8692930

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.

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.

Neuromedin B receptor in esophagus: evidence for subtypes of bombesin receptors

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

Von Schrenck, T.; Heinz-Erian, P.; Moran, T.

1989-04-01

To identify receptors for bombesin-related peptides in the rat esophagus, we measured binding of 125I-Bolton-Hunter neuromedin B (125I-BH-neuromedin B) and 125I-(Tyr4)bombesin to tissue sections from the rat esophagus and compared the results with those for rat pancreas. Esophagus bound both tracers, whereas pancreas bound only 125I-(Tyr4)bombesin. In each tissue binding was saturable, dependent on pH, on time, and on temperature, reversible, and specific. Autoradiography demonstrated binding of both tracers only to the muscularis mucosae of the esophagus and binding of 125I-(Tyr4)bombesin diffusely over pancreatic acini. In the esophagus, the relative potencies for inhibition of binding of both tracers were asmore » follows: neuromedin B greater than bombesin greater than GRP = neuromedin C; similar relative potencies were found for causing contraction of muscle strips from whole esophagus and from the isolated muscularis mucosae. In pancreas tissue sections and dispersed acini, the relative potencies for inhibition of binding of 125I-(Tyr4)bombesin were as follows: bombesin greater than GRP = neuromedin C much greater than neuromedin B. Similar relative potencies were found for stimulation of enzyme secretion from dispersed pancreatic acini. Computer analysis in both tissues demonstrated only a single binding site. The present study demonstrates that rat esophagus muscle possesses specific receptors for bombesin-related peptides. Furthermore, this study shows that the esophageal bombesin receptors represent a previously unidentified class of bombesin receptors in that they have a higher affinity for neuromedin B than for bombesin. In contrast, the pancreatic bombesin receptors have, like all other bombesin receptors described to date, a high affinity for bombesin, but low affinity for neuromedin B.« less

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

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

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

1986-06-01

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

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

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

PubMed Central

Shoelson, S E; Sivaraja, M; Williams, K P; Hu, P; Schlessinger, J; Weiss, M A

1993-01-01

SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism. Images PMID:8382612

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

PubMed Central

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

2014-01-01

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

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

Tactics for preclinical validation of receptor-binding radiotracers

PubMed Central

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

2016-01-01

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

Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation.

PubMed

Grishkovskaya, Irina; Avvakumov, George V; Hammond, Geoffrey L; Catalano, Maria G; Muller, Yves A

2002-08-30

The amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG) contains a single high affinity steroid-binding site. Crystal structures of this domain in complex with several different steroid ligands have revealed that estradiol occupies the SHBG steroid-binding site in an opposite orientation when compared with 5 alpha-dihydrotestosterone or C19 androgen metabolites (5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 beta,17 alpha-diol) or the synthetic progestin levonorgestrel. Substitution of specific residues within the SHBG steroid-binding site confirmed that Ser(42) plays a key role in determining high affinity interactions by hydrogen bonding to functional groups at C3 of the androstanediols and levonorgestrel and the hydroxyl at C17 of estradiol. Among residues participating in the hydrogen bond network with hydroxy groups at C17 of C19 steroids or C3 of estradiol, Asp(65) appears to be the most important. The different binding mode of estradiol is associated with a difference in the position/orientation of residues (Leu(131) and Lys(134)) in the loop segment (Leu(131)-His(136)) that covers the steroid-binding site as well as others (Leu(171)-Lys(173) and Trp(84)) on the surface of human SHBG and may provide a basis for ligand-dependent interactions between SHBG and other macromolecules. These new crystal structures have also enabled us to construct a simple space-filling model that can be used to predict the characteristics of novel SHBG ligands.

Probing the ATP site of GRP78 with nucleotide triphosphate analogs

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

Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun

GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78 ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligandsmore » (ATP analogs) to a receptor (GRP78 ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78 ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg ++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg ++-dependent, as the removal of Mg ++ nearly abolished binding to GRP78 ATPase. The AMPPCP-Mg ++ structure showed evidence for the critical role of Mg ++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg ++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg ++. The 2'-deoxyATP structure showed the conformation of the bound nucleotide flipped out of the active site, explaining the low affinity binding to GRP78 and suggesting that the 2'-OH group is essential for the high affinity binding to GRP78. Altogether, our results demonstrate that GRP78 ATPase possesses nucleotide specificity more relaxed than previously anticipated and can tolerate certain modifications to the nucleobase 7-position and, to a lesser extent, the beta-gamma bridging atom, thereby providing a possible atomic mechanism underlying the transmembrane transport of the ATP analogs.« less

Probing the ATP site of GRP78 with nucleotide triphosphate analogs

DOE PAGES

Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun; ...

2016-05-04

GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78 ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligandsmore » (ATP analogs) to a receptor (GRP78 ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78 ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg ++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg ++-dependent, as the removal of Mg ++ nearly abolished binding to GRP78 ATPase. The AMPPCP-Mg ++ structure showed evidence for the critical role of Mg ++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg ++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg ++. The 2'-deoxyATP structure showed the conformation of the bound nucleotide flipped out of the active site, explaining the low affinity binding to GRP78 and suggesting that the 2'-OH group is essential for the high affinity binding to GRP78. Altogether, our results demonstrate that GRP78 ATPase possesses nucleotide specificity more relaxed than previously anticipated and can tolerate certain modifications to the nucleobase 7-position and, to a lesser extent, the beta-gamma bridging atom, thereby providing a possible atomic mechanism underlying the transmembrane transport of the ATP analogs.« less

Actions of 2-methylpiperidine (MP) and its interactions with (-)-nicotine (N) in the dog and rat

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

Sloan, J.W.; Martin, W.R.; Bostwick, M.

1986-03-01

(+/-)-MP is a highly specific drug for enhancing the binding of (+/-)-(/sup 3/H)N in the rat brain P/sub 2/ preparation. Competition studies employing (+/-)-(/sup 3/H)N as the labelled ligand show that this activity resides in the (+) isomer. Saturation studies employing (+/-)-(/sup 3/H)MP indicate that it binds to a very high affinity site which is presumed to be an up-regulatory site. Studies were conducted where (+/-)-MP was injected through an implanted cannula into the 4th ventricle of intact beagle-type dogs. (+/-)-MP, like low doses of (+)-N, another drug which has specificity in enhancing the binding of (+/-)-, and (-)- andmore » (+)-(/sup 3/H)N, produced EEG synchronization and miosis. (-)-N produced analgesia where as (+/-)-MP produced hyperalgesia. In the urethane-pentobarbital anethetized rats, the i.v. infusion of (+)-MP (600 ..mu..g/kg/min for 10 min), had no effect on heart rate, blood pressure or respiration. Pretreatment with (+)-MP delayed and decreased the bradycardia, vasodepression, and enhanced tidal volume produced by (-)-N infusion (60 ..mu..g/kg/min). These data show that the pharmacologic actions of MP are different from (-)-N and hexamethonium and that MP, which enhances the binding of (+/-)- and (-)-(/sup 3/H)N at the high affinity site, appears to exert opposite effects to and antagonistic effects against (-)-N.« less

Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ

PubMed Central

FAULHAMMER, DIRK; ESCHGFÄLLER, BERND; STARK, SANDRA; BURGSTALLER, PETRA; ENGLBERGER, WERNER; ERFURTH, JEANNETTE; KLEINJUNG, FRANK; RUPP, JOHANNA; VULCU, SEBASTIAN DAN; SCHRÖDER, WERNER; VONHOFF, STEFAN; NAWRATH, HERMANN; GILLEN, CLEMENS; KLUSSMANN, SVEN

2004-01-01

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are l-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to d-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC50 values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPγS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K+ channels. PMID:14970396

Amyloid tracers detect multiple binding sites in Alzheimer's disease brain tissue.

PubMed

Ni, Ruiqing; Gillberg, Per-Göran; Bergfors, Assar; Marutle, Amelia; Nordberg, Agneta

2013-07-01

Imaging fibrillar amyloid-β deposition in the human brain in vivo by positron emission tomography has improved our understanding of the time course of amyloid-β pathology in Alzheimer's disease. The most widely used amyloid-β imaging tracer so far is (11)C-Pittsburgh compound B, a thioflavin derivative but other (11)C- and (18)F-labelled amyloid-β tracers have been studied in patients with Alzheimer's disease and cognitively normal control subjects. However, it has not yet been established whether different amyloid tracers bind to identical sites on amyloid-β fibrils, offering the same ability to detect the regional amyloid-β burden in the brains. In this study, we characterized (3)H-Pittsburgh compound B binding in autopsied brain regions from 23 patients with Alzheimer's disease and 20 control subjects (aged 50 to 88 years). The binding properties of the amyloid tracers FDDNP, AV-45, AV-1 and BF-227 were also compared with those of (3)H-Pittsburgh compound B in the frontal cortices of patients with Alzheimer's disease. Saturation binding studies revealed the presence of high- and low-affinity (3)H-Pittsburgh compound B binding sites in the frontal cortex (K(d1): 3.5 ± 1.6 nM; K(d2): 133 ± 30 nM) and hippocampus (K(d1):5.6 ± 2.2 nM; K(d2): 181 ± 132 nM) of Alzheimer's disease brains. The relative proportion of high-affinity to low-affinity sites was 6:1 in the frontal cortex and 3:1 in the hippocampus. One control showed both high- and low-affinity (3)H-Pittsburgh compound B binding sites (K(d1): 1.6 nM; K(d2): 330 nM) in the cortex while the others only had a low-affinity site (K(d2): 191 ± 70 nM). (3)H-Pittsburgh compound B binding in Alzheimer's disease brains was higher in the frontal and parietal cortices than in the caudate nucleus and hippocampus, and negligible in the cerebellum. Competitive binding studies with (3)H-Pittsburgh compound B in the frontal cortices of Alzheimer's disease brains revealed high- and low-affinity binding sites for BTA-1 (Ki: 0.2 nM, 70 nM), florbetapir (1.8 nM, 53 nM) and florbetaben (1.0 nM, 65 nM). BF-227 displaced 83% of (3)H-Pittsburgh compound B binding, mainly at a low-affinity site (311 nM), whereas FDDNP only partly displaced (40%). We propose a multiple binding site model for the amyloid tracers (binding sites 1, 2 and 3), where AV-45 (florbetapir), AV-1 (florbetaben), and Pittsburgh compound B, all show nanomolar affinity for the high-affinity site (binding site 1), as visualized by positron emission tomography. BF-227 shows mainly binding to site 3 and FDDNP shows only some binding to site 2. Different amyloid tracers may provide new insight into the pathophysiological mechanisms in the progression of Alzheimer's disease.

Surface conformations of an anti-ricin aptamer and its affinity for ricin determined by atomic force microscopy and surface plasmon resonance.

PubMed

Wang, B; Lou, Z; Park, B; Kwon, Y; Zhang, H; Xu, B

2015-01-07

We used atomic force microscopy (AFM) and surface plasmon resonance (SPR) to study the surface conformations of an anti-ricin aptamer and its specific binding affinity for ricin molecules. The effect of surface modification of the Au(111) substrate on the aptamer affinity was also estimated. The AFM topography images had a resolution high enough to distinguish different aptamer conformations. The specific binding site on the aptamer molecule was clearly located by the AFM recognition images. The aptamer on a Au(111) surface modified with carboxymethylated-dextran (CD) showed both similarities to and differences from the one without CD modification. The influence of CD modification was evaluated using AFM images of various aptamer conformations on the Au(111) surface. The affinity between ricin and the anti-ricin aptamer was estimated using the off-rate values measured using AFM and SPR. The SPR measurements of the ricin sample were conducted in the range from 83.3 pM to 8.33 nM, and the limit of detection was estimated as 25 pM (1.5 ng mL(-1)). The off-rate values of the ricin-aptamer interactions were estimated using both single-molecule dynamic force spectroscopy (DFS) and SPR as (7.3 ± 0.4) × 10(-4) s(-1) and (1.82 ± 0.067) × 10(-2) s(-1), respectively. The results show that single-molecule measurements can obtain different reaction parameters from bulk solution measurements. In AFM single-molecule measurements, the various conformations of the aptamer immobilized on the gold surface determined the availability of each specific binding site to the ricin molecules. The SPR bulk solution measurements averaged the signals from specific and non-specific interactions. AFM images and DFS measurements provide more specific information on the interactions of individual aptamer and ricin molecules.

The histidine phosphocarrier protein, HPr, binds to the highly thermostable regulator of sigma D protein, Rsd, and its isolated helical fragments.

PubMed

Neira, José L; Hornos, Felipe; Cozza, Concetta; Cámara-Artigas, Ana; Abián, Olga; Velázquez-Campoy, Adrián

2018-02-01

The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ 70 factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPr sc , and E. coli Rsd, by using fragments derived from each protein. To that end, we used several biophysical probes, namely, fluorescence, CD, NMR, ITC and BLI. Rsd had a free energy of unfolding of 15 kcal mol -1 at 25 °C, and a thermal denaturation midpoint of 103 °C at pH 6.5. The affinity between Rsd and HPr sc was 2 μM. Interestingly enough, the isolated helical-peptides, comprising the third (RsdH3) and fourth (RsdH4) Rsd helices, also interacted with HPr sc in a specific manner, and with affinities similar to that of the whole Rsd. Moreover, the isolated peptide of HPr sc , HPr 9-30 , comprising the active site, His15, also was bound to intact Rsd with similar affinity. Therefore, binding between Rsd and HPr sc was modulated by the two helices H3 and H4 of Rsd, and the regions around the active site of HPr sc . This implies that specific fragments of Rsd and HPr sc can be used to interfere with other protein-protein interactions (PPIs) of each other protein. Copyright © 2018 Elsevier Inc. All rights reserved.

Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides.

PubMed

Zhang, Pengzhi; Tripathi, Swarnendu; Trinh, Hoa; Cheung, Margaret S

2017-03-28

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca 2+ ) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca 2+ /CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca 2+ signaling pathways in neurons. It was shown experimentally that Ca 2+ /CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca 2+ -free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca 2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng 13-49 ), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng 13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng 13-49 structures by matching the changes in the chemical shifts of CaM upon Ng 13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca 2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca 2+ for CaM in the presence of Ng 13-49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca 2+ binding loops particularly at C-domain of CaM, enabling Ca 2+ release. In contrast, CaMKII peptide increases Ca 2+ affinity for the C-domain of CaM by stabilizing the two Ca 2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng 13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca 2+ binding affinities. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2016-01-01

Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently available approaches and provides a new route to design tailored and well-controlled hybrid nanoparticles. PMID:27622533

Functional somatostatin receptors on a rat pancreatic acinar cell line

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

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

1988-07-01

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

[18F]F15599, a novel 5-HT1A receptor agonist, as a radioligand for PET neuroimaging.

PubMed

Lemoine, Laëtitia; Verdurand, Mathieu; Vacher, Bernard; Blanc, Elodie; Le Bars, Didier; Newman-Tancredi, Adrian; Zimmer, Luc

2010-03-01

The serotonin-1A (5-HT(1A)) receptor is implicated in the pathophysiology of major neuropsychiatric disorders. Thus, the functional imaging of 5-HT(1A) receptors by positron emission tomography (PET) may contribute to the understanding of its role in those pathologies and their therapeutics. These receptors exist in high- and low-affinity states and it is proposed that agonists bind preferentially to the high-affinity state of the receptor and therefore could provide a measure of the functional 5-HT(1A) receptors. Since all clinical PET 5-HT(1A) radiopharmaceuticals are antagonists, it is of great interest to develop a( 18)F labelled agonist. F15599 (3-chloro-4-fluorophenyl-(4-fluoro-4{[(5-methyl-pyrimidin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl)-methanone) is a novel ligand with high affinity and selectivity for 5-HT(1A) receptors and is currently tested as an antidepressant. In pharmacological tests in rat, it exhibits preferential agonist activity at post-synaptic 5-HT(1A) receptors in cortical brain regions. Here, its nitro-precursor was synthesised and radiolabelled via a fluoronucleophilic substitution. Radiopharmacological evaluations included in vitro and ex vivo autoradiography in rat brain and PET scans on rats and cats. Results were compared with simultaneous studies using [(18)F]MPPF, a validated 5-HT(1A) antagonist radiopharmaceutical. The chemical and radiochemical purities of [(18)F]F15599 were >98%. In vitro [(18)F]F15599 binding was consistent with the known 5-HT(1A) receptors distribution (hippocampus, dorsal raphe nucleus, and notably cortical areas) and addition of Gpp(NH)p inhibited [(18)F]F15599 binding, consistent with a specific binding to G protein-coupled receptors. In vitro binding of [(18)F]F15599 was blocked by WAY100635 and 8-OH-DPAT, respectively, prototypical 5-HT(1A) antagonist and agonist. The ex vivo and in vivo studies demonstrated that the radiotracer readily entered the rat and the cat brain and generated few brain radioactive metabolites. Remarkably, in microPET studies, [(18)F]F15599 notably displayed a pattern of brain labelling that did not correlate with in vitro observations. Thus, in cat, the highest binding was observed in dorsal raphe and cingulate cortex with little binding in other cortical regions and none in hippocampus. In vivo binding was abolished by WAY100635, indicating specific labelling of 5-HT(1A) receptors. [(18)F]F15599 is a radiofluorinated agonist presenting interesting characteristics for probing in vitro and in vivo the high-affinity states of the 5-HT(1A) receptors. Its differential labelling of 5-HT(1A) receptors in vitro and in vivo may result from its reported preferential interaction with receptors coupled to specific G-protein subtypes.

Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

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

Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F.

2008-08-19

Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to themore » lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.« less

Effect of N-benzoyl-D-phenylalanine and metformin on insulin receptors in neonatal streptozotocin-induced diabetic rats: studies on insulin binding to erythrocytes.

PubMed

Ashokkumar, N; Pari, L; Rao, Ch Appa

2006-07-01

In the present study, we focused on the insulin-receptor binding in circulating erythrocytes of N-benzoyl-D-phenylalanine (NBDP) and metformin in neonatal streptozotocin (nSTZ)-induced male Wistar rats. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors in NBDP and metformin-treated diabetic rats. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (53.0 +/- 3.1%) than in NBDP (62.0 +/- 3.1%), metformin (66.0 +/- 3.3%) and NBDP and metformin combination-treated (72.0 +/- 4.2%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with NBDP and metformin-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from NBDP and metformin-treated diabetic rats having NBDP 2.0 +/- 0.10 x 10(-10) M(-1) (Kd1); 12.0 +/- 0.85 x 10(-8) M(-1) (Kd2), Metformin 2.1 +/- 0.15 x 10(-10) M(-1) (Kd1); 15.0 +/- 0.80 x 10(-8) M(-1) (Kd2), NBDP and metformin 2.7 +/- 0.10 x 10(-10) M(-1) (Kd1); 20.0 +/- 1.2 x 10(-8) M(-1) (Kd2) compared with 0.9 +/- 0.06 x 10(-10) M(-1) (Kd1); 6.0 +/- 0.30 x 10(-8) M(-1) (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in nSTZ induced diabetic control rats. Treatment with NBDP along with metformin significantly improved specific insulin binding, with receptor number and affinity binding reaching almost normal non-diabetic levels. The data presented here show that NBDP along with metformin increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin.

Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by 15N and 2H ESEEM Spectroscopy

PubMed Central

Vogt, Matthew; Lahiri, Simanti; Hoogstraten, Charles G.; Britt, R. David; DeRose, Victoria J.

2010-01-01

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a Kd,app < 10 µM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin–echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands. PMID:17177426

RNA Modulates the Interaction between Influenza A Virus NS1 and Human PABP1.

PubMed

Arias-Mireles, Bryan H; de Rozieres, Cyrus M; Ly, Kevin; Joseph, Simpson

2018-05-25

Nonstructural protein 1 (NS1) is a multifunctional protein involved in preventing host-interferon response in influenza A virus (IAV). Previous studies have indicated that NS1 also stimulates the translation of viral mRNA by binding to conserved sequences in the viral 5'-UTR. Additionally, NS1 binds to poly(A) binding protein 1 (PABP1) and eukaryotic initiation factor 4G (eIF4G). The interaction of NS1 with the viral 5'-UTR, PABP1, and eIF4G has been suggested to specifically enhance the translation of viral mRNAs. In contrast, we report that NS1 does not directly bind to sequences in the viral 5'-UTR, indicating that NS1 is not responsible for providing the specificity to stimulate viral mRNA translation. We also monitored the interaction of NS1 with PABP1 using a new, quantitative FRET assay. Our data show that NS1 binds to PABP1 with high affinity; however, the binding of double-stranded RNA (dsRNA) to NS1 weakens the binding of NS1 to PABP1. Correspondingly, the binding of PABP1 to NS1 weakens the binding of NS1 to double-stranded RNA (dsRNA). In contrast, the affinity of PABP1 for binding to poly(A) RNA is not significantly changed by NS1. We propose that the modulation of NS1·PABP1 interaction by dsRNA may be important for the viral cycle.

Characterization, solubilization and partial purification of serotonin 5-HT1C receptors

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

Yagaloff, K.A.

1986-01-01

/sup 125/I-Lysergic acid diethylamide (/sup 125/I-LSD) binds with high affinity to a unique serotonergic site on rat choroid plexus. These sites were localized to choroid plexus epithelial cells using a novel high resolution autoradiographic technique. In membrane preparations, the serotonergic site density was 3100 fmol/mg protein, which is 10 fold higher than the density of any other serotonergic site in brain homogenates. The pharmacology of this site, termed the 5-HT1c site, does not match that of 5-Ht1a, 5-HT1b or 5HT2 serotonergic sites. 5-Ht1c sites were solubilized from pig choroid plexus using the zwitterionic detergent, CHAPS. High affinity labelling of themore » solubilized site was obtained using the serotonergic radioligand, N1-methyl-2-(/sup 125/I)lysergic acid diethylamide (/sup 125/I-MIL). Choroid plexus tumors obtained from transgenic mice were examined for the presence of serotonin 5-HT1c receptors. /sup 125/I-LSD binding to choroid plexus tumors displays a pharmacological profile that matches the properties of 5-HT1c receptors in normal choroid plexus. The tumor exhibits the highest site density of serotonin receptors (6600 fmol/mg protein) found in any tissue. /sup 125/I-LSD autoradiography of brain sections from transgenic mice shows high levels of specific labelling over the tumor. The affinities of various indolealkyl, phenlakyl and beta-carboline derivatives for the serotonin 5-HT1c receptor were measured in pig choroid plexus using /sup 125/I-MIL. Serotonin precursors and metabolites were all very weak inhibitors of specific /sup 125/I-MIL binding. Structure-affinity relationships were determined for a number of indolealkylamine analogues. Only serotonin is present in cerebrospinal fluid at concentrations near its 5-HT1c inhibition constant, suggesting that serotonin is the natural 5-HT1c agonist.« less

Analysis of the Glutamate Agonist LY404,039 Binding to Nonstatic Dopamine Receptor D2 Dimer Structures and Consensus Docking.

PubMed

Salmas, Ramin Ekhteiari; Seeman, Philip; Aksoydan, Busecan; Erol, Ismail; Kantarcioglu, Isik; Stein, Matthias; Yurtsever, Mine; Durdagi, Serdar

2017-06-21

Dopamine receptor D2 (D2R) plays an important role in the human central nervous system and is a focal target of antipsychotic agents. The D2 High R and D2 Low R dimeric models previously developed by our group are used to investigate the prediction of binding affinity of the LY404,039 ligand and its binding mechanism within the catalytic domain. The computational data obtained using molecular dynamics simulations fit well with the experimental results. The calculated binding affinities of LY404,039 using MM/PBSA for the D2 High R and D2 Low R targets were -12.04 and -9.11 kcal/mol, respectively. The experimental results suggest that LY404,039 binds to D2 High R and D2 Low R with binding affinities (K i ) of 8.2 and 1640 nM, respectively. The high binding affinity of LY404,039 in terms of binding to [ 3 H]domperidone was inhibited by the presence of a guanine nucleotide, indicating an agonist action of the drug at D2 High R. The interaction analysis demonstrated that while Asp114 was among the most critical amino acids for D2 High R binding, residues Ser193 and Ser197 were significantly more important within the binding cavity of D2 Low R. Molecular modeling analyses are extended to ensemble docking as well as structure-based pharmacophore model (E-pharmacophore) development using the bioactive conformation of LY404,039 at the binding pocket as a template and screening of small-molecule databases with derived pharmacophore models.

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

PubMed Central

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

2012-01-01

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

Zn(II) and Hg(II) binding to a designed peptide that accommodates different coordination geometries.

PubMed

Szunyogh, Dániel; Gyurcsik, Béla; Larsen, Flemming H; Stachura, Monika; Thulstrup, Peter W; Hemmingsen, Lars; Jancsó, Attila

2015-07-28

Designed metal ion binding peptides offer a variety of applications in both basic science as model systems of more complex metalloproteins, and in biotechnology, e.g. in bioremediation of toxic metal ions, biomining or as artificial enzymes. In this work a peptide (HS: Ac-SCHGDQGSDCSI-NH2) has been specifically designed for binding of both Zn(II) and Hg(II), i.e. metal ions with different preferences in terms of coordination number, coordination geometry, and to some extent ligand composition. It is demonstrated that HS accommodates both metal ions, and the first coordination sphere, metal ion exchange between peptides, and speciation are characterized as a function of pH using UV-absorption-, synchrotron radiation CD-, (1)H-NMR-, and PAC-spectroscopy as well as potentiometry. Hg(II) binds to the peptide with very high affinity in a {HgS2} coordination geometry, bringing together the two cysteinates close to each end of the peptide in a loop structure. Despite the high affinity, Hg(II) is kinetically labile, exchanging between peptides on the subsecond timescale, as indicated by line broadening in (1)H-NMR. The Zn(II)-HS system displays more complex speciation, involving monomeric species with coordinating cysteinates, histidine, and a solvent water molecule, as well as HS-Zn(II)-HS complexes. In summary, the HS peptide displays conformational flexibility, contains many typical metal ion binding groups, and is able to accommodate metal ions with different structural and ligand preferences with high affinity. As such, the HS peptide may be a scaffold offering binding of a variety of metal ions, and potentially serve for metal ion sequestration in biotechnological applications.

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

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.

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

Wang, Wei; Liu, Yang; Zu, Xiangyang

Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection.more » A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.« less

Synthetic Fab Fragments that Bind the HIV-1 gp41 Heptad Repeat Regions

PubMed Central

Liu, Yanyun; Regula, Lauren K.; Stewart, Alex; Lai, Jonathan R.

2011-01-01

Recent work has demonstrated that antibody phage display libraries containing restricted diversity in the complementarity determining regions (CDRs) can be used to target a wide variety of antigens with high affinity and specificity. In the most extreme case, antibodies whose combining sites are comprised of only two residues – tyrosine and serine – have been identified against several protein antigens. [F. A. Fellouse, B. Li, D. M. Compaan, A. A. Peden, S. G. Hymowitz, and S. S. Sidhu, J. Mol. Biol., 348 (2005) 1153–1162.] Here, we report the isolation and characterization of antigen-binding fragments (Fabs) from such “minimalist” diversity synthetic antibody libraries that bind the heptad repeat regions of human immunodeficiency virus type 1 (HIV-1) gp41. We show that these Fabs are highly specific for the HIV-1 epitope and comparable in affinity to a single chain variable fragment (scFv) derived from a natural antibody repertoire that targets the same region. Since the heptad repeat regions of HIV-1 gp41 are required for viral entry, these Fabs have potential for use in therapeutic, research, or diagnostic applications. PMID:21925149

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

PubMed

Endo, T; Ohbayashi, H; Kanazawa, K; Kochibe, N; Kobata, A

1992-01-15

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.

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

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

PubMed

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

2018-04-02

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

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

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

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

1985-08-01

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

Simultaneous prediction of binding free energy and specificity for PDZ domain-peptide interactions

NASA Astrophysics Data System (ADS)

Crivelli, Joseph J.; Lemmon, Gordon; Kaufmann, Kristian W.; Meiler, Jens

2013-12-01

Interactions between protein domains and linear peptides underlie many biological processes. Among these interactions, the recognition of C-terminal peptides by PDZ domains is one of the most ubiquitous. In this work, we present a mathematical model for PDZ domain-peptide interactions capable of predicting both affinity and specificity of binding based on X-ray crystal structures and comparative modeling with R osetta. We developed our mathematical model using a large phage display dataset describing binding specificity for a wild type PDZ domain and 91 single mutants, as well as binding affinity data for a wild type PDZ domain binding to 28 different peptides. Structural refinement was carried out through several R osetta protocols, the most accurate of which included flexible peptide docking and several iterations of side chain repacking and backbone minimization. Our findings emphasize the importance of backbone flexibility and the energetic contributions of side chain-side chain hydrogen bonds in accurately predicting interactions. We also determined that predicting PDZ domain-peptide interactions became increasingly challenging as the length of the peptide increased in the N-terminal direction. In the training dataset, predicted binding energies correlated with those derived through calorimetry and specificity switches introduced through single mutations at interface positions were recapitulated. In independent tests, our best performing protocol was capable of predicting dissociation constants well within one order of magnitude of the experimental values and specificity profiles at the level of accuracy of previous studies. To our knowledge, this approach represents the first integrated protocol for predicting both affinity and specificity for PDZ domain-peptide interactions.

High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems.

PubMed

Tremblay, Reynald; Feng, Mary; Menassa, Rima; Huner, Norman P A; Jevnikar, Anthony M; Ma, Shengwu

2011-04-01

Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. SBA has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. The success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant SBA (rSBA). Here, we demonstrate the utility of transient and stable expression systems in Nicotiana benthamiana and potato, respectively, for the production of rSBA, with the transgenic protein accumulated to 4% of total soluble protein (TSP) in Nicotiana benthamiana leaves and 0.3% of TSP in potato tubers. Furthermore, we show that both plant-derived rSBAs retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native SBA, retained their binding specificity for N-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. Affinity column purification using N-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rSBA. This work is the first step toward use of rSBA for various new applications, including the development of rSBA as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs.

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning*

PubMed Central

van Rosmalen, Martijn; Janssen, Brian M. G.; Hendrikse, Natalie M.; van der Linden, Ardjan J.; Pieters, Pascal A.; Wanders, Dave; de Greef, Tom F. A.; Merkx, Maarten

2017-01-01

Meditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging, and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning. Deep sequencing was used to construct a fitness landscape of this protein-peptide interaction, and four mutations were identified that together improved the affinity for cetuximab 10-fold to 15 nm. Importantly, the increased affinity translated into enhanced cetuximab-mediated recruitment to EGF receptor-overexpressing cancer cells. Although in silico Rosetta simulations correctly identified positions that were tolerant to mutation, modeling did not accurately predict the affinity-enhancing mutations. The experimental approach reported here should be generally applicable and could be used to develop meditope peptides with low nanomolar affinity for other therapeutic antibodies. PMID:27974464

Polyvalent immunoglobulin binding is an obstacle to accurate measurement of specific antibodies with ELISA despite inclusion of blocking agents.

PubMed

Loeffler, David A; Klaver, Andrea C

2017-11-01

Specific antibody concentrations are frequently measured in serum (and plasma and intravenous immunoglobulin) samples by enzyme-linked immunosorbent assay (ELISA). The standard negative control involves incubation of buffer alone on antigen-coated wells. The immunoreactivity that develops in antigen-coated wells in which diluted serum has been incubated is assumed to represent specific antibody binding. This approach can result in marked overestimation of specific antibody levels, because serum contains specific polyvalent antibodies which bind, primarily with low affinity, to multiple antigens (including those on ELISA plates) despite the use of blocking agents. Non-denaturing purification of serum IgG, followed by assessment of the antigen binding or antigen-binding affinity of this purified IgG, can reduce but not eliminate the problem of polyvalent antibody binding in indirect ELISAs. Alternatively, polyvalent antibody binding can be estimated by incubating a diluted serum sample on wells coated with an irrelevant protein (such as bovine serum albumin or a scrambled peptide sequence) or buffer alone, then subtracting this reactivity from the sample's binding to wells coated with the antigen of interest. Polyvalent binding of immunoglobulins must be accounted for in order to obtain accurate ELISA measurements of serum, plasma, or intravenous immunoglobulin antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

Ion-binding properties of Calnuc, Ca2+ versus Mg2+--Calnuc adopts additional and unusual Ca2+-binding sites upon interaction with G-protein.

PubMed

Kanuru, Madhavi; Samuel, Jebakumar J; Balivada, Lavanya M; Aradhyam, Gopala K

2009-05-01

Calnuc is a novel, highly modular, EF-hand containing, Ca(2+)-binding, Golgi resident protein whose functions are not clear. Using amino acid sequences, we demonstrate that Calnuc is a highly conserved protein among various organisms, from Ciona intestinalis to humans. Maximum homology among all sequences is found in the region that binds to G-proteins. In humans, it is known to be expressed in a variety of tissues, and it interacts with several important protein partners. Among other proteins, Calnuc is known to interact with heterotrimeric G-proteins, specifically with the alpha-subunit. Herein, we report the structural implications of Ca(2+) and Mg(2+) binding, and illustrate that Calnuc functions as a downstream effector for G-protein alpha-subunit. Our results show that Ca(2+) binds with an affinity of 7 mum and causes structural changes. Although Mg(2+) binds to Calnuc with very weak affinity, the structural changes that it causes are further enhanced by Ca(2+) binding. Furthermore, isothermal titration calorimetry results show that Calnuc and the G-protein bind with an affinity of 13 nm. We also predict a probable function for Calnuc, that of maintaining Ca(2+) homeostasis in the cell. Using Stains-all and terbium as Ca(2+) mimic probes, we demonstrate that the Ca(2+)-binding ability of Calnuc is governed by the activity-based conformational state of the G-protein. We propose that Calnuc adopts structural sites similar to the ones seen in proteins such as annexins, c2 domains or chromogrannin A, and therefore binds more calcium ions upon binding to Gialpha. With the number of organelle-targeted G-protein-coupled receptors increasing, intracellular communication mediated by G-proteins could become a new paradigm. In this regard, we propose that Calnuc could be involved in the downstream signaling of G-proteins.

Structures of the Streptococcus sanguinis SrpA Binding Region with Human Sialoglycans Suggest Features of the Physiological Ligand.

PubMed

Loukachevitch, Lioudmila V; Bensing, Barbara A; Yu, Hai; Zeng, Jie; Chen, Xi; Sullam, Paul M; Iverson, T M

2016-10-11

Streptococcus sanguinis is a leading cause of bacterial infective endocarditis, a life-threatening infection of heart valves. S. sanguinis binds to human platelets with high avidity, and this adherence is likely to enhance virulence. Previous studies suggest that a serine-rich repeat adhesin termed SrpA mediates the binding of S. sanguinis to human platelets via its interaction with sialoglycans on the receptor GPIbα. However, in vitro binding assays with SrpA and defined sialoglycans failed to identify specific high-affinity ligands. To improve our understanding of the interaction between SrpA and human platelets, we determined cocrystal structures of the SrpA sialoglycan binding region (SrpA BR ) with five low-affinity ligands: three sialylated trisaccharides (sialyl-T antigen, 3'-sialyllactose, and 3'-sialyl-N-acetyllactosamine), a sialylated tetrasaccharide (sialyl-Lewis X ), and a sialyl galactose disaccharide component common to these sialoglyans. We then combined structural analysis with mutagenesis to further determine whether our observed interactions between SrpA BR and glycans are important for binding to platelets and to better map the binding site for the physiological receptor. We found that the sialoglycan binding site of SrpA BR is significantly larger than the sialoglycans cocrystallized in this study, which suggests that binding of SrpA to platelets either is multivalent or occurs via a larger, disialylated glycan.

Structures of the Streptococcus sanguinis SrpA Binding Region with Human Sialoglycans Suggest Features of the Physiological Ligand

PubMed Central

Loukachevitch, Lioudmila V.; Bensing, Barbara A.; Yu, Hai; Zeng, Jie; Chen, Xi; Sullam, Paul M.; Iverson, T M

2016-01-01

Streptococcus sanguinis is a leading cause of bacterial infective endocarditis, a life threatening infection of heart valves. S. sanguinis binds to human platelets with high avidity, and this adherence is likely to enhance virulence. Previous studies suggest that a serine-rich repeat adhesin termed SrpA mediates the binding of S. sanguinis to human platelets via its interaction with sialoglycans on the receptor GPIbα. However, in vitro binding assays between SrpA and defined sialoglycans failed to identify specific high-affinity ligands. To better understand the interaction between SrpA and human platelets, we determined cocrystal structures of the SrpA sialoglycan binding region (SrpABR) with five low-affinity ligands: three sialylated trisaccharides (sialyl-T antigen, 3'-sialyllactose, and 3'-sialyl-N-acetyllactosamine), a sialylated tetrasaccharide (sialyl-LewisX) and a sialyl galactose disaccharide component common to these sialoglyans. We then combined structural analysis with mutagenesis to further determine whether our observed interactions between SrpABR and glycans are important for binding to platelets and to better map the binding site for the physiological receptor. We found that the sialoglycan binding site of SrpABR is significantly larger than the sialoglycans cocrystallized in this study, which suggests that SrpA binding to platelets is either multivalent or occurs via a larger, disialylated glycan. PMID:27685666

Aptamer-based liposomes improve specific drug loading and release.

PubMed

Plourde, Kevin; Derbali, Rabeb Mouna; Desrosiers, Arnaud; Dubath, Céline; Vallée-Bélisle, Alexis; Leblond, Jeanne

2017-04-10

Aptamer technology has shown much promise in cancer therapeutics for its targeting abilities. However, its potential to improve drug loading and release from nanocarriers has not been thoroughly explored. In this study, we employed drug-binding aptamers to actively load drugs into liposomes. We designed a series of DNA aptamer sequences specific to doxorubicin, displaying multiple binding sites and various binding affinities. The binding ability of aptamers was preserved when incorporated into cationic liposomes, binding up to 15equivalents of doxorubicin per aptamer, therefore drawing the drug into liposomes. Optimization of the charge and drug/aptamer ratios resulted in ≥80% encapsulation efficiency of doxorubicin, ten times higher than classical passively-encapsulating liposomal formulations and similar to a pH-gradient active loading strategy. In addition, kinetic release profiles and cytotoxicity assay on HeLa cells demonstrated that the release and therapeutic efficacy of liposomal doxorubicin could be controlled by the aptamer's structure. Our results suggest that the aptamer exhibiting a specific intermediate affinity is the best suited to achieve high drug loading while maintaining efficient drug release and therapeutic activity. This strategy was successfully applied to tobramycin, a hydrophilic drug suffering from low encapsulation into liposomes, where its loading was improved six-fold using aptamers. Overall, we demonstrate that aptamers could act, in addition to their targeting properties, as multifunctional excipients for liposomal formulations. Copyright © 2017 Elsevier B.V. All rights reserved.

Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

PubMed

Clark, Andrew G; Naufer, M Nabuan; Westerlund, Fredrik; Lincoln, Per; Rouzina, Ioulia; Paramanathan, Thayaparan; Williams, Mark C

2018-02-06

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

2016-03-01

Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

Imaging of Cerebral Amyloid Angiopathy with Bivalent 99mTc-Hydroxamamide Complexes

NASA Astrophysics Data System (ADS)

Iikuni, Shimpei; Ono, Masahiro; Watanabe, Hiroyuki; Matsumura, Kenji; Yoshimura, Masashi; Kimura, Hiroyuki; Ishibashi-Ueda, Hatsue; Okamoto, Yoko; Ihara, Masafumi; Saji, Hideo

2016-05-01

Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer’s disease (AD). We previously reported 99mTc-hydroxamamide (99mTc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for β-amyloid peptide (Aβ(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aβ(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for 99mTc-Ham complexes, and all 99mTc-Ham complexes with an amyloid ligand showed binding affinity for Aβ(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent 99mTc-Ham complexes ([99mTc]SB2A and [99mTc]BT2B), and they displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the 99mTc-Ham complex.

Imaging of Cerebral Amyloid Angiopathy with Bivalent (99m)Tc-Hydroxamamide Complexes.

PubMed

Iikuni, Shimpei; Ono, Masahiro; Watanabe, Hiroyuki; Matsumura, Kenji; Yoshimura, Masashi; Kimura, Hiroyuki; Ishibashi-Ueda, Hatsue; Okamoto, Yoko; Ihara, Masafumi; Saji, Hideo

2016-05-16

Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer's disease (AD). We previously reported (99m)Tc-hydroxamamide ((99m)Tc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for β-amyloid peptide (Aβ(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aβ(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for (99m)Tc-Ham complexes, and all (99m)Tc-Ham complexes with an amyloid ligand showed binding affinity for Aβ(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent (99m)Tc-Ham complexes ([(99m)Tc]SB2A and [(99m)Tc]BT2B), and they displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the (99m)Tc-Ham complex.

Staphylokinase has distinct modes of interaction with antimicrobial peptides, modulating its plasminogen-activation properties

PubMed Central

Nguyen, Leonard T.; Vogel, Hans J.

2016-01-01

Staphylokinase (Sak) is a plasminogen activator protein that is secreted by many Staphylococcus aureus strains. Sak also offers protection by binding and inhibiting specific antimicrobial peptides (AMPs). Here, we evaluate Sak as a more general interaction partner for AMPs. Studies with melittin, mCRAMP, tritrpticin and bovine lactoferricin indicate that the truncation of the first ten residues of Sak (SakΔN10), which occurs in vivo and uncovers important residues in a bulge region, improves its affinity for AMPs. Melittin and mCRAMP have a lower affinity for SakΔN10, and in docking studies, they bind to the N-terminal segment and bulge region of SakΔN10. By comparison, lactoferricin and tritrpticin form moderately high affinity 1:1 complexes with SakΔN10 and their cationic residues form several electrostatic interactions with the protein’s α-helix. Overall, our work identifies two distinct AMP binding surfaces on SakΔN10 whose occupation would lead to either inhibition or promotion of its plasminogen activating properties. PMID:27554435

The structural basis for function in diamond-like carbon binding peptides.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Linder, Markus B

2014-07-29

The molecular structural basis for the function of specific peptides that bind to diamond-like carbon (DLC) surfaces was investigated. For this, a competition assay that provided a robust way of comparing relative affinities of peptide variants was set up. Point mutations of specific residues resulted in significant effects, but it was shown that the chemical composition of the peptide was not sufficient to explain peptide affinity. More significantly, rearrangements in the sequence indicated that the binding is a complex recognition event that is dependent on the overall structure of the peptide. The work demonstrates the unique properties of peptides for creating functionality at interfaces via noncovalent binding for potential applications in, for example, nanomaterials, biomedical materials, and sensors.

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.

Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

NASA Technical Reports Server (NTRS)

Knisley, Keith A.; Rodkey, L. Scott

1986-01-01

A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

In silico maturation of binding-specificity of DNA aptamers against Proteus mirabilis.

PubMed

Savory, Nasa; Lednor, Danielle; Tsukakoshi, Kaori; Abe, Koichi; Yoshida, Wataru; Ferri, Stefano; Jones, Brian V; Ikebukuro, Kazunori

2013-10-01

Proteus mirabilis is a prominent cause of catheter-associated urinary tract infections (CAUTIs) among patients undergoing long-term bladder catheterization. There are currently no effective means of preventing P. mirabilis infections, and strategies for prophylaxis and rapid early diagnosis are urgently required. Aptamers offer significant potential for development of countermeasures against P. mirabilis CAUTI and are an ideal class of molecules for the development of diagnostics and therapeutics. Here we demonstrate the application of Cell-SELEX to identify DNA aptamers that show high affinity for P. mirabilis. While the aptamers identified displayed high affinity for P. mirabilis cells in dot blotting assays, they also bound to other uropathogenic bacteria. To improve aptamer specificity for P. mirabilis, an in silico maturation (ISM) approach was employed. Two cycles of ISM allowed the identification of an aptamer showing 36% higher specificity, evaluated as a ratio of binding signal for P. mirabilis to that for Escherichia coli (also a cause of CAUTI and the most common urinary tract pathogen). Aptamers that specifically recognize P. mirabilis would have diagnostic and therapeutic values and constitute useful tools for studying membrane-associated proteins in this organism. Copyright © 2013 Wiley Periodicals, Inc.

Developing tools for risk assessment in protected species: relative potencies inferred from competitive binding of halogenated aromatic hydrocarbons to aryl hydrocarbon receptors from beluga (Delphinapterus leucas) and mouse

PubMed Central

Jensen, Brenda A.; Reddy, Christopher M.; Nelson, Robert K.; Hahn, Mark E.

2011-01-01

Persistent organic pollutants such as halogenated aromatic hydrocarbons (HAHs) biomagnify in food webs and accumulate to high concentrations in top predators like odontocete cetaceans (toothed whales). The most toxic HAHs are the 2,3,7,8-substituted halogenated dibenzo-p-dioxins and furans, and non-ortho-substituted polychlorinated biphenyls (PCBs), which exert their effects via the aryl hydrocarbon receptor (AHR). Understanding the impact of HAHs in wildlife is limited by the lack of taxon-specific information about the relative potencies of toxicologically important congeners. To assess whether Toxic Equivalency Factors (TEFs) determined in rodents are predictive of HAH relative potencies in a cetacean, we used beluga and mouse AHRs expressed in vitro from cloned cDNAs to measure the relative AHR-binding affinities of ten HAHs from five different structural classes. The rank order of mean IC50s for competitive binding to beluga AHR was: TCDD

Developing tools for risk assessment in protected species: Relative potencies inferred from competitive binding of halogenated aromatic hydrocarbons to aryl hydrocarbon receptors from beluga (Delphinapterus leucas) and mouse.

PubMed

Jensen, Brenda A; Reddy, Christopher M; Nelson, Robert K; Hahn, Mark E

2010-11-01

Persistent organic pollutants such as halogenated aromatic hydrocarbons (HAHs) biomagnify in food webs and accumulate to high concentrations in top predators like odontocete cetaceans (toothed whales). The most toxic HAHs are the 2,3,7,8-substituted halogenated dibenzo-p-dioxins and furans, and non-ortho-substituted polychlorinated biphenyls (PCBs), which exert their effects via the aryl hydrocarbon receptor (AHR). Understanding the impact of HAHs in wildlife is limited by the lack of taxon-specific information about the relative potencies of toxicologically important congeners. To assess whether Toxic Equivalency Factors (TEFs) determined in rodents are predictive of HAH relative potencies in a cetacean, we used beluga and mouse AHRs expressed in vitro from cloned cDNAs to measure the relative AHR-binding affinities of ten HAHs from five different structural classes. The rank order of mean IC(50)s for competitive binding to beluga AHR was: TCDD

Closely Related Antibody Receptors Exploit Fundamentally Different Strategies for Steroid Recognition

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

Verdino, P.; Aldag, C.; Hilvert, D.

2009-05-26

Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu{sup H47}Trp and Arg{sup H100}Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determinedmore » the crystal structures of the 1E9 Leu{sup H47}Trp/Arg{sup H100}Trp double mutant (1E9dm) as an unliganded Fab at 2.05 {angstrom} resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 {angstrom}. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.« less

Oxytocin and vasopressin: distinct receptors in myometrium

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

Guillon, G.; Balestre, M.N.; Roberts, J.M.

1987-06-01

The binding characteristics of (/sup 3/H)oxytocin (( /sup 3/H)OT) and (/sup 3/H)lysine vasopressin (( /sup 3/H)LVP) to nonpregnant human myometrium were investigated. Binding of both radioligands was saturable, time dependent, and reversible. Whereas (/sup 3/H)OT was found to bind to a single class of sites with high affinity (Kd, 1.5 +/- 0.4 (+/- SEM) nM) and low capacity (maximum binding (Bmax), 34 +/- 6 fmol/mg protein), (/sup 3/H)LVP bound to two classes of sites, one with high affinity (Kd, 2.2 +/- 0.1 nM) and low capacity (Bmax, 198 +/- 7 fmol/mg protein) and another with low affinity (Kd, 655 +/-more » 209 nM) and high capacity (Bmax, 5794 +/- 1616 fmol/mg protein). The binding of the labeled peptides also displayed a marked difference in sensitivity to Mg2+ and guanine nucleotides. These differences in binding characteristics as well as the differences in potency of analogs in competing for (/sup 3/H)OT and (/sup 3/H)LVP binding indicate the presence of distinct receptors for OT and vasopressin in human myometrium. Pharmacological characterization of the high affinity binding sites for (/sup 3/H)LVP indicated that these are of the V1 subtype. Although, as suggested by others, vasopressin and OT can bind to the same sites, the presence of distinct receptors for both peptides provides an explanation for the previously reported difference in myometrial responsiveness to OT and vasopressin.« less

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

Bidlack, J.M.; Frey, D.K.; Seyed-Mozaffari, A.

The binding properties of 14{beta}-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM ({sup 3}H)(D-Ala{sup 2},(Me)Phe{sup 4},Gly(ol){supmore » 5})enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the {mu} binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The {mu} receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the {delta}-selective peptide ({sup 3}H)(D-penicillamine{sup 2},D-penicillamine{sup 5})enkephalin (DPDPE) and (-)-({sup 3}H)bremazocine in the presence of {mu} and {delta} blockers, selective for {kappa} binding sites. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the {mu}s site did not afford protection. These studies have demonstrated that when a disulfide bond at or near {mu} opioid binding sites was reduced, BAM could then alkylate this site, resulting in the specific irreversible labeling of {mu} opioid receptors.« less

Alterations in Hemagglutinin Receptor-Binding Specificity Accompany the Emergence of Highly Pathogenic Avian Influenza Viruses

PubMed Central

Mochalova, Larisa; Harder, Timm; Tuzikov, Alexander; Bovin, Nicolai; Wolff, Thorsten; Matrosovich, Mikhail; Schweiger, Brunhilde

2015-01-01

ABSTRACT Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galβ1-4GlcNAcβ (3′SLN) and Neu5Acα2-3Galβ1-3GlcNAcβ (SiaLec) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3′SLN and Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ (SiaLex), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors. IMPORTANCE Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical “poultry-like” receptor, 3′SLN, is provided by substitutions in the receptor-binding site of HA which appeared in HA of LPAIVs in the course of transmission of LPAIVs from wild waterfowl into poultry flocks, with subsequent adaptation in poultry. The identification of LPAIVs with receptor characteristics of HPAIVs argues that the sialic acid-binding specificity of the HA may be used as a novel phenotypic marker of HPAIVs. PMID:25741006

Metal loading levels influence on REE distribution on humic acid: Experimental and Modelling approach

NASA Astrophysics Data System (ADS)

Marsac, R.; Davranche, M.; Gruau, G.; Dia, A.

2009-04-01

In natural organic-rich waters, rare earth elements (REE) speciation is mainly controlled by organic colloids such as humic acid (HA). Different series of REE-HA complexation experiments performed at several metal loading (REE/C) displayed two pattern shapes (i) at high metal loading, a middle-REE (MREE) downward concavity, and (ii) at low metal loading, a regular increase from La to Lu (e.g. Sonke and Salters, 2006; Pourret et al., 2007). Both REE patterns might be related to REE binding with different surface sites on HA. To understand REE-HA binding, REE-HA complexation experiments at various metals loading were carried out using ultrafiltration combined with ICP-MS measurements, for the 14 REE simultaneously. The patterns of the apparent coefficients of REE partition between HA and the inorganic solution (log Kd) evolved regularly according to the metal loading. The REE patterns presented a MREE downward concavity at low loading and a regular increase from La to Lu at high loading. The dataset was modelled with Model VI by adjusting two specific parameters, log KMA, the apparent complexation constant of HA low affinity sites and DLK2, the parameter increasing high affinity sites binding strength. Experiments and modelling provided evidence that HA high affinity sites controlled the REE binding with HA at low metal loading. The REE-HA complex could be as multidentate complexes with carboxylic or phenolic sites or potentially with sites constituted of N, P or S as donor atoms. Moreover, these high affinity sites could be different for light and heavy REE, because heavy REE have higher affinity for these sites, in low density, and could saturate them. These new Model VI parameter sets allowed the prediction of the REE-HA pattern shape evolution on a large range of pH and metal loading. According to the metal loading, the evolution of the calculated REE patterns was similar to the various REE pattern observed in natural acidic organic-rich waters (pH<7 and DOC>10 mg L-1). As a consequence, the metal loading could be the key parameter controlling the REE pattern in organic-rich waters.

Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein.

PubMed

Zhang, Zhen; Palzkill, Timothy

2003-11-14

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

[Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

PubMed

Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

2012-01-01

It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein.

PubMed

Meng, Q; Li, M; Silberg, M A; Conrad, F; Bettencourt, J; To, R; Huang, C; Ma, J; Meyer, K; Shimizu, R; Cao, L; Tomic, M T; Marks, J D

2012-02-15

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development, including pharmacokinetic (PK), toxicology, stability, and biochemical characterization studies of such drugs. We have developed an antitoxin, XOMA 3AB, consisting of three recombinant mAbs that potently neutralize the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind nonoverlapping BoNT/A epitopes with high affinity. XOMA 3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. mAb-specific domains were used to develop an enzyme-linked immunosorbent assay (ELISA) for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay that is robust to interference from components in serum was also developed, and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that binds the same protein and is superior to anti-idiotype approaches. Copyright © 2011 Elsevier Inc. All rights reserved.

Domain based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein

PubMed Central

Meng, Q.; Li, M.; Silberg, M.A.; Conrad, F.; Bettencourt, J.; To, R.; Huang, C.; Ma, J.; Meyer, K.; Shimizu, R.; Cao, L.; Tomic, M.T.; Marks, J.D.

2014-01-01

Quantitation of individual mAbs within a combined antibody drug product is required for preclinical and clinical drug development including pharmacokinetics (PK), toxicology, stability and biochemical characterization studies of such drugs. We have developed an antitoxin (XOMA 3AB) consisting of three recombinant monoclonal antibodies (mAbs) that potently neutralizes the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind non-overlapping BoNT/A epitopes with high affinity. XOMA3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from E. coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. MAb specific domains were used to develop an ELISA for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay was also developed that is robust to interference from components in serum and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein and is superior to anti-idiotype approaches. PMID:22037290

Functionalized poly(ethylene glycol) diacrylate microgels by microfluidics: In situ peptide encapsulation for in serum selective protein detection.

PubMed

Celetti, Giorgia; Natale, Concetta Di; Causa, Filippo; Battista, Edmondo; Netti, Paolo A

2016-09-01

Polymeric microparticles represent a robustly platform for the detection of clinically relevant analytes in biological samples; they can be functionalized encapsulating a multiple types of biologics entities, enhancing their applications as a new class of colloid materials. Microfluidic offers a versatile platform for the synthesis of monodisperse and engineered microparticles. In this work, we report microfluidic synthesis of novel polymeric microparticles endowed with specific peptide due to its superior specificity for target binding in complex media. A peptide sequence was efficiently encapsulated into the polymeric network and protein binding occurred with high affinity (KD 0.1-0.4μM). Fluidic dynamics simulation was performed to optimize the production conditions for monodisperse and stable functionalized microgels. The results demonstrate the easy and fast realization, in a single step, of functionalized monodisperse microgels using droplet-microfluidic technique, and how the inclusion of the peptide within polymeric network improve both the affinity and the specificity of protein capture. Copyright © 2016 Elsevier B.V. All rights reserved.

Specific binding of [(18)F]fluoroethyl-harmol to monoamine oxidase A in rat brain cryostat sections, and compartmental analysis of binding in living brain.

PubMed

Maschauer, Simone; Haller, Adelina; Riss, Patrick J; Kuwert, Torsten; Prante, Olaf; Cumming, Paul

2015-12-01

We investigated [(18)F]fluoroethyl-harmol ([(18)F]FEH) as a reversible and selective ligand for positron emission tomography (PET) studies of monoamine oxidase A (MAO-A). Binding of [(18)F]FEH in rat brain cryostat sections indicated high affinity (KD = 3 nM), and density (Bmax; 600 pmol/g). The plasma free fraction was 45%, and untransformed parent constituted only 13% of plasma radioactivity at 10 min after injection. Compartmental analysis of PET recordings in pargyline-treated rats showed high permeability to brain (K1; 0.32 mL/g/min) and slow washout (k2; 0.024/min), resulting in a uniformly high equilibrium distribution volume (VD; 20 mL/g). Using this VD to estimate unbound ligand in brain of untreated rats, the binding potential ranged from 4.2 in cerebellum to 7.2 in thalamus. We also calculated maps of rats receiving [(18)F]FEH at a range of specific activities, and then estimated saturation binding parameters in the living brain. In thalamus, striatum and frontal cortex KD was globally close to 300 nM and Bmax was close to 1600 pmol/g; the 100-fold discrepancy in affinity suggests a very low free fraction for [(18)F]FEH in the living brain. Based on a synthesis of findings, we calculate the endogenous dopamine concentration to be 0.4 μM in the striatal compartment containing MAO-A, thus unlikely to exert competition against [(18)F]FEH binding in vivo. In summary, [(18)F]FEH has good properties for the detection of MAO-A in the rat brain by PET, and may present logistic advantages for clinical research at centers lacking a medical cyclotron. We made a compartmental analysis of [(18)F]fluoroethylharmol ([(18)F]FEH) binding to monoamine oxidase A (MAO-A) in living rat brain and estimated the saturation binding parameters from the binding potential (BPND). The Bmax was of comparable magnitude to that in vitro, but with apparent affinity (300 nM), it was 100-fold lower in vivo. PET imaging with [(18) F]FEH is well suited for quantitation of MAO-A in living brain. © 2015 International Society for Neurochemistry.

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

PubMed

Birchfield, N B; Casida, J E

1996-01-01

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

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

PubMed

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

2005-04-01

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

Engineering of the function of diamond-like carbon binding peptides through structural design.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Singh, Vivek K; Mikkilä, Joona; Kostiainen, Mauri A; Koskinen, Jari; Linder, Markus B

2015-02-09

The use of phage display to select material-specific peptides provides a general route towards modification and functionalization of surfaces and interfaces. However, a rational structural engineering of the peptides for optimal affinity is typically not feasible because of insufficient structure-function understanding. Here, we investigate the influence of multivalency of diamond-like carbon (DLC) binding peptides on binding characteristics. We show that facile linking of peptides together using different lengths of spacers and multivalency leads to a tuning of affinity and kinetics. Notably, increased length of spacers in divalent systems led to significantly increased affinities. Making multimers influenced also kinetic aspects of surface competition. Additionally, the multivalent peptides were applied as surface functionalization components for a colloidal form of DLC. The work suggests the use of a set of linking systems to screen parameters for functional optimization of selected material-specific peptides.

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

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

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

PubMed Central

Naveau, Marie; Lazennec-Schurdevin, Christine; Panvert, Michel; Dubiez, Etienne; Mechulam, Yves; Schmitt, Emmanuelle

2013-01-01

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex. PMID:23193270

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

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

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF ormore » thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).« less

Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

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

Earle, M.; Concas, A.; Yamamura, H.I.

1986-03-01

The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. atmore » 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.« less

Surface-modified multifunctional MIP nanoparticles

NASA Astrophysics Data System (ADS)

Moczko, Ewa; Poma, Alessandro; Guerreiro, Antonio; Perez de Vargas Sansalvador, Isabel; Caygill, Sarah; Canfarotta, Francesco; Whitcombe, Michael J.; Piletsky, Sergey

2013-04-01

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors.The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors. Electronic supplementary information (ESI) available: Details of the synthesis of eosin O-acrylate monomer and 1H-NMR spectrum of MIP NPs post-derivatised with PEG shell. See DOI: 10.1039/c3nr00354j

Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

PubMed Central

Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

2016-01-01

Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

Decorin is a Zn(2+) Metalloprotein

NASA Technical Reports Server (NTRS)

Yang, Vivian W.-C.; LaBrenz, Steven R.; Rosenberg, Lawrence C.; McQuillan, David; Hoeoek, Magnus

1998-01-01

Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. We here demonstrate that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells, has a high affinity for Zn(2+). Binding of Zn(2+) to decorin is demonstrated by Zn(2+) chelating chromatography and equilibrium dialyses. The Zn(2+) binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in the spacing reminiscent of a Zn finger. A recombinant 41 amino acid long peptide representing the N-terminal domain of decorin has full Zn(2+) binding activity and binds two Zn(2+) ions with an average K(D) of 3 x 10(exp -7) M. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn(2+) binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, did not bind Zn(2+) with high affinity.

Psathyrella velutina Mushroom Lectin Exhibits High Affinity toward Sialoglycoproteins Possessing Terminal N-Acetylneuraminic Acid alpha 2,3-Linked to Penultimate Galactose Residues of Trisialyl N-Glycans. Comparison with other sialic acid-specific lectins.

PubMed

Ueda, Haruko; Matsumoto, Hanako; Takahashi, Noriko; Ogawa, Haruko

2002-07-12

A lectin from the fruiting body of the Psathyrella velutina mushroom (PVL) was found to bind specifically to N-acetylneuraminic acid, as well as to GlcNAc (Ueda, H., Kojima, K., Saitoh, T., and Ogawa, H. (1999) FEBS Lett. 448, 75-80). In this study, the glycan sequences that PVL recognizes with high affinity on sialoglycoproteins were revealed. Among sialic acid-specific lectins only PVL could reveal the sialylated N-acetyllactosamine structure of glycoproteins in blotting studies, based on the dual specificity. The affinity of PVL to fetuin was measured by surface plasmon resonance to be 10(7) m(-1), which is an order of magnitude higher than those of Sambucus nigra agglutinin and Maackia amurensis mitogen, whereas affinity to asialofetuin was approximately 0 and to asialo-agalactofetuin was 10(8) m(-1), suggesting that PVL exhibits remarkably high affinities toward glycoproteins possessing trisialo- or GlcNAc-exposed glycans. Transferrin was separated into fractions that correspond to the sialylation states on an immobilized PVL column. Transferrin-possessing trisialoglycans containing alpha2,3-linked N-acetylneuraminic acid on the beta1,4-linked GlcNAc branch bound to the PVL column and eluted with GlcNAc; those containing only alpha2,6-linked sialic acids were retarded, whereas other transferrin fractions passed through the column. These results indicate that PVL is a lectin with potential for separation and detection of sialoglycoproteins because of its dual specificity toward sialoglycans and GlcNAc exposed glycans.

Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom.

PubMed

Kochibe, N; Matta, K L

1989-01-05

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.

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

PHOENIX: a scoring function for affinity prediction derived using high-resolution crystal structures and calorimetry measurements.

PubMed

Tang, Yat T; Marshall, Garland R

2011-02-28

Binding affinity prediction is one of the most critical components to computer-aided structure-based drug design. Despite advances in first-principle methods for predicting binding affinity, empirical scoring functions that are fast and only relatively accurate are still widely used in structure-based drug design. With the increasing availability of X-ray crystallographic structures in the Protein Data Bank and continuing application of biophysical methods such as isothermal titration calorimetry to measure thermodynamic parameters contributing to binding free energy, sufficient experimental data exists that scoring functions can now be derived by separating enthalpic (ΔH) and entropic (TΔS) contributions to binding free energy (ΔG). PHOENIX, a scoring function to predict binding affinities of protein-ligand complexes, utilizes the increasing availability of experimental data to improve binding affinity predictions by the following: model training and testing using high-resolution crystallographic data to minimize structural noise, independent models of enthalpic and entropic contributions fitted to thermodynamic parameters assumed to be thermodynamically biased to calculate binding free energy, use of shape and volume descriptors to better capture entropic contributions. A set of 42 descriptors and 112 protein-ligand complexes were used to derive functions using partial least-squares for change of enthalpy (ΔH) and change of entropy (TΔS) to calculate change of binding free energy (ΔG), resulting in a predictive r2 (r(pred)2) of 0.55 and a standard error (SE) of 1.34 kcal/mol. External validation using the 2009 version of the PDBbind "refined set" (n = 1612) resulted in a Pearson correlation coefficient (R(p)) of 0.575 and a mean error (ME) of 1.41 pK(d). Enthalpy and entropy predictions were of limited accuracy individually. However, their difference resulted in a relatively accurate binding free energy. While the development of an accurate and applicable scoring function was an objective of this study, the main focus was evaluation of the use of high-resolution X-ray crystal structures with high-quality thermodynamic parameters from isothermal titration calorimetry for scoring function development. With the increasing application of structure-based methods in molecular design, this study suggests that using high-resolution crystal structures, separating enthalpy and entropy contributions to binding free energy, and including descriptors to better capture entropic contributions may prove to be effective strategies toward rapid and accurate calculation of binding affinity.

Imino proton exchange rates imply an induced-fit binding mechanism for the VEGF165-targeting aptamer, Macugen

PubMed Central

Lee, Joon-Hwa; Jucker, Fiona; Pardi, Arthur

2008-01-01

The 2′-fluoro/2′-O-methyl modified RNA aptamer Macugen is a potent inhibitor of the angiogenic regulatory protein, VEGF165. Macugen binds with high affinity to the heparin-binding domain (HBD) of VEGF165. Hydrogen exchange rates of the imino protons were measured for free Macugen and Macugen bound to the HBD or full-length VEGF to better understand the mechanism for high affinity binding. The results here show that the internal loop and hairpin loop of Macugen are highly dynamic in the free state and are greatly stabilized and/or protected from solvent upon protein binding. PMID:18485899

Takifugu rubripes cation independent mannose 6-phosphate receptor: Cloning, expression and functional characterization of the IGF-II binding domain.

PubMed

A, Ajith Kumar; Nadimpalli, Siva Kumar

2018-07-01

Mannose 6-phosphate/IGF-II receptor mediated lysosomal clearance of insulin-like growth factor-II is significantly associated with the evolution of placental mammals. The protein is also referred to as the IGF-II receptor. Earlier studies suggested relatively low binding affinity between the receptor and ligand in prototherian and metatherian mammals. In the present study, we cloned the IGF-II binding domain of the early vertebrate fugu fish and expressed it in bacteria. A 72000Da truncated receptor containing the IGF-II binding domain was obtained. Analysis of this protein (covering domains 11-13 of the CIMPR) for its affinity to fish and human IGF-II by ligand blot assays and ELISA showed that the expressed receptor can specifically bind to both fish and human IGF-II. Additionally, a peptide-specific antibody raised against the region of the IGF-II binding domain also was able to recognize the IGF-II binding regions of mammalian and non-mammalian cation independent MPR protein. These interactions were further characterized by Surface Plasma resonance support that the receptor binds to fish IGF-II, with a dissociation constant of 548nM. Preliminary analysis suggests that the binding mechanism as well as the affinity of the fish and human receptor for IGF-II may have varied according to different evolutionary pressures. Copyright © 2018. Published by Elsevier B.V.

Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

PubMed

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

PubMed Central

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible. PMID:23874971

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.

Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

PubMed

Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

2004-09-01

We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

PubMed Central

David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick

2018-01-01

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998

Morph-X-Select: Morphology-based tissue aptamer selection for ovarian cancer biomarker discovery

PubMed Central

Wang, Hongyu; Li, Xin; Volk, David E.; Lokesh, Ganesh L.-R.; Elizondo-Riojas, Miguel-Angel; Li, Li; Nick, Alpa M.; Sood, Anil K.; Rosenblatt, Kevin P.; Gorenstein, David G.

2016-01-01

High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5′dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of human ovarian tissue and human microvascular endothelial cells. This new Morph-X-Select method allows us to select high-affinity aptamers and their associated target proteins in a specific and accurate way, and could be used for personalized biomarker discovery to improve medical decision-making and to facilitate the development of targeted therapies to achieve more favorable outcomes. PMID:27839510

Influence of quasi-specific sites on kinetics of target DNA search by a sequence-specific DNA-binding protein.

PubMed

Kemme, Catherine A; Esadze, Alexandre; Iwahara, Junji

2015-11-10

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such "quasi-specific" sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1's association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins.

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.

Structural Basis for High Specificity of Amadori Compound and Mannopine Opine Binding in Bacterial Pathogens*

PubMed Central

Marty, Loïc; Vigouroux, Armelle; Aumont-Nicaise, Magali; Dessaux, Yves; Faure, Denis; Moréra, Solange

2016-01-01

Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars. They are Agrobacterium nutrients and imported into the bacterial cell via periplasmic-binding proteins (PBPs) and ABC-transporters. Mannopine, an opine from the mannityl-opine family, is synthesized from an intermediate named deoxy-fructosyl-glutamine (DFG), which is also an opine and abundant Amadori compound (a name used for any derivative of aminodeoxysugars) present in decaying plant materials. The PBP MotA is responsible for mannopine import in mannopine-assimilating agrobacteria. In the nopaline-opine type agrobacteria strain, SocA protein was proposed as a putative mannopine binding PBP, and AttC protein was annotated as a mannopine binding-like PBP. Structural data on mannityl-opine-PBP complexes is currently lacking. By combining affinity data with analysis of seven x-ray structures at high resolution, we investigated the molecular basis of MotA, SocA, and AttC interactions with mannopine and its DFG precursor. Our work demonstrates that AttC is not a mannopine-binding protein and reveals a specific binding pocket for DFG in SocA with an affinity in nanomolar range. Hence, mannopine would not be imported into nopaline-type agrobacteria strains. In contrast, MotA binds both mannopine and DFG. We thus defined one mannopine and two DFG binding signatures. Unlike mannopine-PBPs, selective DFG-PBPs are present in a wide diversity of bacteria, including Actinobacteria, α-,β-, and γ-proteobacteria, revealing a common role of this Amadori compound in pathogenic, symbiotic, and opportunistic bacteria. PMID:27609514

Electrostatic Interactions Mediate Binding of Obscurin to Small Ankyrin 1: Biochemical and Molecular Modeling Studies

PubMed Central

Busby, Ben; Oashi, Taiji; Willis, Chris D.; Ackermann, Maegen A.; Kontrogianni-Konstantopoulos, Aikaterini; MacKerell, Alexander D.; Bloch, Robert J.

2012-01-01

Small ankyrin 1 (sAnk1; also Ank1.5) is an integral protein of the sarcoplasmic reticulum in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro, in combination with site directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for obscurin consists in part of six lysine and arginine residues. Here we show that four charged residues in the high affinity binding site on obscurin for sAnk1, between residues 6316-6345, consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on obscurin to bind with high affinity to sAnk1. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in obscurin. We used molecular modeling to develop structural models of residues 6322-6339 of obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for obscurin is organized as two ankyrin-like repeats, with the last α-helical segment oriented at an angle to the nearby helices, allowing lysine-6338 of obscurin to form an ionic interaction with aspartate-111 of sAnk1. This prediction was validated by double mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on obscurin and sAnk1 promote binding and complex formation. PMID:21333652

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.