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Sample records for protein binding microarrays

  1. Quantifying the Antibody Binding on Protein Microarrays using Microarray Nonlinear Calibration

    PubMed Central

    Yu, Xiaobo; Wallstrom, Garrick; Magee, Dewey Mitchell; Qiu, Ji; Mendoza, D. Eliseo A.; Wang, Jie; Bian, Xiaofang; Graves, Morgan; LaBaer, Joshua

    2015-01-01

    To address the issue of quantification for antibody assays with protein microarrays, we firstly developed a Microarray Nonlinear Calibration (MiNC) method that applies in the quantification of antibody binding to the surface of microarray spots. We found that MiNC significantly increased the linear dynamic range and reduced assay variations. A serological analysis of guinea pig Mycobacterium tuberculosis models showed that a larger number of putative antigen targets were identified with MiNC, which is consistent with the improved assay performance of protein microarrays. We expect that our cumulative results will provide scientists with a new appreciation of antibody assays with protein microarrays. Our MiNC method has the potential to be employed in biomedical research with multiplex antibody assays which need quantitation, including the discovery of antibody biomarkers, clinical diagnostics with multi-antibody signatures and construction of immune mathematical models. PMID:23662896

  2. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  3. Using protein-binding microarrays to study transcription factor specificity: homologs, isoforms and complexes

    PubMed Central

    Andrilenas, Kellen K.; Penvose, Ashley

    2015-01-01

    Protein–DNA binding is central to specificity in gene regulation, and methods for characterizing transcription factor (TF)–DNA binding remain crucial to studies of regulatory specificity. High-throughput (HT) technologies have revolutionized our ability to characterize protein–DNA binding by significantly increasing the number of binding measurements that can be performed. Protein-binding microarrays (PBMs) are a robust and powerful HT platform for studying DNA-binding specificity of TFs. Analysis of PBM-determined DNA-binding profiles has provided new insight into the scope and mechanisms of TF binding diversity. In this review, we focus specifically on the PBM technique and discuss its application to the study of TF specificity, in particular, the binding diversity of TF homologs and multi-protein complexes. PMID:25431149

  4. Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

    NASA Astrophysics Data System (ADS)

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins.

  5. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures. PMID:26039143

  6. Detection of the specific binding on protein microarrays by oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Lu, Heng; Wen, Juan; Wang, Xu; Yuan, Kun; Li, Wei; Lu, Huibin; Zhou, Yueliang; Jin, Kuijuan; Ruan, Kangcheng; Yang, Guozhen

    2010-09-01

    The specific binding between Cy5-labeled goat anti-mouse Immunoglobulin G (IgG) and mouse IgG with a concentration range from 625 to 104 µg ml - 1 has been detected successfully by the oblique-incidence reflectivity difference (OI-RD) method in each procedure of microarray fabrication. The experimental data prove that the OI-RD method can be employed not only to distinguish the different concentrations in label-free fashion but also to detect the antibody-antigen capture. In addition, the differential treatment of the OI-RD signals can decrease the negative influences of glass slide as the microarray upholder. Therefore the OI-RD technique has promising applications for the label-free and high-throughput detection of protein microarrays.

  7. Screening of the binding of small molecules to proteins by desorption electrospray ionization mass spectrometry combined with protein microarray.

    PubMed

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ. PMID:26174365

  8. Host Tissue and Glycan Binding Specificities of Avian Viral Attachment Proteins Using Novel Avian Tissue Microarrays

    PubMed Central

    Ambepitiya Wickramasinghe, Iresha N.; de Vries, Robert P.; Eggert, Amber M.; Wandee, Nantaporn; de Haan, Cornelis A. M.; Gröne, Andrea; Verheije, Monique H.

    2015-01-01

    The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens. PMID:26035584

  9. Boosting AthaMap Database Content with Data from Protein Binding Microarrays.

    PubMed

    Hehl, Reinhard; Norval, Leo; Romanov, Artyom; Bülow, Lorenz

    2016-01-01

    The AthaMap database generates a map of predicted transcription factor binding sites (TFBS) and small RNA target sites for the whole Arabidopsis thaliana genome. With the advent of protein binding microarrays (PBM), the number of known TFBS for A. thaliana transcription factors (TFs) has increased dramatically. Using 113 positional weight matrices (PWMs) generated from a single PBM study and representing a total number of 68 different TFs, the number of predicted TFBS in AthaMap was now increased by about 3.8 × 10(7) to 4.9 × 10(7). The number of TFs with PWM-predicted TFBS annotated in AthaMap has increased to 126, representing a total of 29 TF families and newly including ARF, AT-Hook, YABBY, LOB/AS2 and SRS. Furthermore, links from all Arabidopsis TFs and genes to the newly established Arabidopsis Information Portal (AIP) have been implemented. With this qualitative and quantitative update, the improved AthaMap increases its value as a resource for the analysis of A. thaliana gene expression regulation at www.athamap.de. PMID:26542109

  10. Self-Assembling Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ramachandran, Niroshan; Hainsworth, Eugenie; Bhullar, Bhupinder; Eisenstein, Samuel; Rosen, Benjamin; Lau, Albert Y.; C. Walter, Johannes; LaBaer, Joshua

    2004-07-01

    Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

  11. Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening.

    PubMed

    Han, Jin-Hee; Li, Jian; Wang, Bo; Lee, Seong-Kyun; Nyunt, Myat Htut; Na, Sunghun; Park, Jeong-Hyun; Han, Eun-Taek

    2015-08-01

    Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown. PMID:26323838

  12. Measurement of Small Molecule Binding Kinetics on a Protein Microarray by Plasmonic-Based Electrochemical Impedance Imaging

    PubMed Central

    2015-01-01

    We report on a quantitative study of small molecule binding kinetics on protein microarrays with plasmonic-based electrochemical impedance microscopy (P-EIM). P-EIM measures electrical impedance optically with high spatial resolution by converting a surface charge change to a surface plasmon resonance (SPR) image intensity change, and the signal is not scaled to the mass of the analyte. Using P-EIM, we measured binding kinetics and affinity between small molecule drugs (imatinib and SB202190) and their target proteins (kinases Abl1 and p38-α). The measured affinity values are consistent with reported values measured by an indirect competitive binding assay. We also found that SB202190 has weak bindings to ABL1 with KD > 10 μM, which is not reported in the literature. Furthermore, we found that P-EIM is less prone to nonspecific binding, a long-standing issue in SPR. Our results show that P-EIM is a novel method for high-throughput measurement of small molecule binding kinetics and affinity, which is critical to the understanding of small molecules in biological systems and discovery of small molecule drugs. PMID:25153794

  13. Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality.

    PubMed

    Lei, Zhen; Gao, Jiaxue; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-04-27

    We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL(-1) for MMP-2 and 5.7 pg mL(-1) for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained. PMID:27049528

  14. Immunoprofiling Using NAPPA Protein Microarrays

    PubMed Central

    Sibani, Sahar; LaBaer, Joshua

    2012-01-01

    Protein microarrays provide an efficient method to immunoprofile patients in an effort to rapidly identify disease immunosignatures. The validity of using autoantibodies in diagnosis has been demonstrated in type 1 diabetes, rheumatoid arthritis, and systemic lupus, and is now being strongly considered in cancer. Several types of protein microarrays exist including antibody and antigen arrays. In this chapter, we describe the immunoprofiling application for one type of antigen array called NAPPA (nucleic acids programmable protein array). We provide a guideline for setting up the screening study and designing protein arrays to maximize the likelihood of obtaining quality data. PMID:21370064

  15. High-throughput allogeneic antibody detection using protein microarrays.

    PubMed

    Paul, Jed; Sahaf, Bita; Perloff, Spenser; Schoenrock, Kelsi; Wu, Fang; Nakasone, Hideki; Coller, John; Miklos, David

    2016-05-01

    Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used to detect alloantibodies in patient plasma samples post hematopoietic cell transplantation (HCT); however, protein microarrays have the potential to be multiplexed, more sensitive, and higher throughput than ELISAs. Here, we describe the development of a novel and sensitive microarray method for detection of allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome, called HY antigens. Six microarray surfaces were tested for their ability to bind recombinant protein and peptide HY antigens. Significant allogeneic immune responses were determined in male patients with female donors by considering normal male donor responses as baseline. HY microarray results were also compared with our previous ELISA results. Our overall goal was to maximize antibody detection for both recombinant protein and peptide epitopes. For detection of HY antigens, the Epoxy (Schott) protein microarray surface was both most sensitive and reliable and has become the standard surface in our microarray platform. PMID:26902899

  16. Protein microarrays as tools for functional proteomics.

    PubMed

    LaBaer, Joshua; Ramachandran, Niroshan

    2005-02-01

    Protein microarrays present an innovative and versatile approach to study protein abundance and function at an unprecedented scale. Given the chemical and structural complexity of the proteome, the development of protein microarrays has been challenging. Despite these challenges there has been a marked increase in the use of protein microarrays to map interactions of proteins with various other molecules, and to identify potential disease biomarkers, especially in the area of cancer biology. In this review, we discuss some of the promising advances made in the development and use of protein microarrays. PMID:15701447

  17. Applications in high-content functional protein microarrays.

    PubMed

    Moore, Cedric D; Ajala, Olutobi Z; Zhu, Heng

    2016-02-01

    Protein microarray technology provides a versatile platform for characterization of hundreds to thousands of proteins in a parallel and high-throughput manner. Over the last decade, applications of functional protein microarrays in particular have flourished in studying protein function at a systems level and have led to the construction of networks and pathways describing these functions. Relevant areas of research include the detection of various binding properties of proteins, the study of enzyme-substrate relationships, the analysis of host-microbe interactions, and profiling antibody specificity. In addition, discovery of novel biomarkers in autoimmune diseases and cancers is emerging as a major clinical application of functional protein microarrays. In this review, we will summarize the recent advances of functional protein microarrays in both basic and clinical applications. PMID:26599287

  18. A protein multiplex microarray substrate with high sensitivity and specificity

    PubMed Central

    Fici, Dolores A.; McCormick, William; Brown, David W.; Herrmann, John E.; Kumar, Vikram; Awdeh, Zuheir L.

    2010-01-01

    The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specificory signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laborat environment. PMID:20974147

  19. A Protein Microarray ELISA for Screening Biological Fluids

    SciTech Connect

    Varnum, Susan M.; Woodbury, Ronald L.; Zangar, Richard C.

    2004-02-01

    Protein microarrays permit the simultaneous measurement of many proteins in a small sample volume and therefore provide an attractive approach for the quantitative measurement of proteins in biological fluids, including serum. This chapter describes a microarray ELISA assay. Capture antibodies are immobilized onto a glass surface, the covalently attached antibodies bind a specific antigen from a sample overlaying the array. A second, biotinylated antibody that recognizes the same antigen as the first antibody but at a different epitope is then used for detection. Detection is based upon an enzymatic signal enhancement method known as tyramide signal amplification (TSA). By coupling a microarray-ELISA format with the signal amplification of tyramide deposition, the assay sensitivity is as low as sub-pg/ml.

  20. Protein microarrays for parasite antigen discovery.

    PubMed

    Driguez, Patrick; Doolan, Denise L; Molina, Douglas M; Loukas, Alex; Trieu, Angela; Felgner, Phil L; McManus, Donald P

    2015-01-01

    The host serological profile to a parasitic infection, such as schistosomiasis, can be used to define potential vaccine and diagnostic targets. Determining the host antibody response using traditional approaches is hindered by the large number of putative antigens in any parasite proteome. Parasite protein microarrays offer the potential for a high-throughput host antibody screen to simplify this task. In order to construct the array, parasite proteins are selected from available genomic sequence and protein databases using bioinformatic tools. Selected open reading frames are PCR amplified, incorporated into a vector for cell-free protein expression, and printed robotically onto glass slides. The protein microarrays can be probed with antisera from infected/immune animals or humans and the antibody reactivity measured with fluorophore labeled antibodies on a confocal laser microarray scanner to identify potential targets for diagnosis or therapeutic or prophylactic intervention. PMID:25388117

  1. Protein-Based Microarray for the Detection of Pathogenic Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarrays have been used for gene expression and protein interaction studies, but recently, multianalyte diagnostic assays have employed the microarray platform. We developed a microarray immunoassay for bacteria, with biotinylated capture antibodies on streptavidin slides. To complete the fluor...

  2. Protein microarray: sensitive and effective immunodetection for drug residues

    PubMed Central

    2010-01-01

    Background Veterinary drugs such as clenbuterol (CL) and sulfamethazine (SM2) are low molecular weight (<1000 Da) compounds, or haptens, that are difficult to develop immunoassays due to their low immunogenicity. In this study, we conjugated the drugs to ovalbumin to increase their immunogenicity for antiserum production in rabbits and developed a protein microarray immunoassay for detection of clenbuterol and sulfamethazine. The sensitivity of this approach was then compared to traditional ELISA technique. Results The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA) showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g) than the ci-ELISA (0.1 ng/g) for detection of CL residues. Conclusions The protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique. PMID:20158905

  3. Applications of protein microarrays for biomarker discovery

    PubMed Central

    Ramachandran, Niroshan; Srivastava, Sanjeeva; LaBaer, Joshua

    2011-01-01

    The search for new biomarkers for diagnosis, prognosis and therapeutic monitoring of diseases continues in earnest despite dwindling success at finding novel reliable markers. Some of the current markers in clinical use do not provide optimal sensitivity and specificity, with the prostate cancer antigen (PSA) being one of many such examples. The emergence of proteomic techniques and systems approaches to study disease pathophysiology has rekindled the quest for new biomarkers. In particular the use of protein microarrays has surged as a powerful tool for large scale testing of biological samples. Approximately half the reports on protein microarrays have been published in the last two years especially in the area of biomarker discovery. In this review, we will discuss the application of protein microarray technologies that offer unique opportunities to find novel biomarkers. PMID:21136793

  4. Plant protein kinase substrates identification using protein microarrays.

    PubMed

    Ma, Shisong; Dinesh-Kumar, Savithramma P

    2015-01-01

    Protein kinases regulate signaling pathways by phosphorylating their targets. They play critical roles in plant signaling networks. Although many important protein kinases have been identified in plants, their substrates are largely unknown. We have developed and produced plant protein microarrays with more than 15,000 purified plant proteins. Here, we describe a detailed protocol to use these microarrays to identify plant protein kinase substrates via in vitro phosphorylation assays on these arrays. PMID:25930701

  5. Quantifying protein–protein interactions in high throughput using protein domain microarrays

    PubMed Central

    Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

    2011-01-01

    Protein microarrays provide an efficient way to identify and quantify protein–protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (KDs) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein–ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein–protein interaction networks. PMID:20360771

  6. Protein Microarrays for the Detection of Biothreats

    NASA Astrophysics Data System (ADS)

    Herr, Amy E.

    Although protein microarrays have proven to be an important tool in proteomics research, the technology is emerging as useful for public health and defense applications. Recent progress in the measurement and characterization of biothreat agents is reviewed in this chapter. Details concerning validation of various protein microarray formats, from contact-printed sandwich assays to supported lipid bilayers, are presented. The reviewed technologies have important implications for in vitro characterization of toxin-ligand interactions, serotyping of bacteria, screening of potential biothreat inhibitors, and as core components of biosensors, among others, research and engineering applications.

  7. Microarray-based technologies for the discovery of selective, RNA-binding molecules.

    PubMed

    Abulwerdi, Fardokht A; Schneekloth, John S

    2016-07-01

    The identification of small molecules that bind specifically to RNA is a challenge. However, the recent explosion in knowledge about the role RNA plays in a number of physiological processes apart from coding for protein sequences makes it a highly interesting target for chemical probes and therapeutics. One technology that has played an important role in the discovery of RNA-binding molecules is microarrays. Microarrays have been broadly employed to screen, profile, and quantify RNA interactions, and will likely play an important role in the discovery of new classes of ligands going forward. Here, we discuss the development of microarray technologies, including aminoglycoside, peptide, peptoid, and small molecule microarrays, and their use in studying RNA-interacting molecules. PMID:27109057

  8. Data Analysis Strategies for Protein Microarrays

    PubMed Central

    Díez, Paula; Dasilva, Noelia; González-González, María; Matarraz, Sergio; Casado-Vela, Juan; Orfao, Alberto; Fuentes, Manuel

    2012-01-01

    Microarrays constitute a new platform which allows the discovery and characterization of proteins. According to different features, such as content, surface or detection system, there are many types of protein microarrays which can be applied for the identification of disease biomarkers and the characterization of protein expression patterns. However, the analysis and interpretation of the amount of information generated by microarrays remain a challenge. Further data analysis strategies are essential to obtain representative and reproducible results. Therefore, the experimental design is key, since the number of samples and dyes, among others aspects, would define the appropriate analysis method to be used. In this sense, several algorithms have been proposed so far to overcome analytical difficulties derived from fluorescence overlapping and/or background noise. Each kind of microarray is developed to fulfill a specific purpose. Therefore, the selection of appropriate analytical and data analysis strategies is crucial to achieve successful biological conclusions. In the present review, we focus on current algorithms and main strategies for data interpretation.

  9. Probing small molecule microarrays with tagged proteins in cell lysates

    PubMed Central

    Pop, Marius S.; Wassaf, Dina; Koehler, Angela N.

    2014-01-01

    The technique of small-molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal-to-noise ratios (SNR). SMM screening is a high throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding-based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment. PMID:25445177

  10. Protein Microarrays--Without a Trace

    SciTech Connect

    Camarero, J A

    2007-04-05

    Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.

  11. Advancing translational research with next-generation protein microarrays.

    PubMed

    Yu, Xiaobo; Petritis, Brianne; LaBaer, Joshua

    2016-04-01

    Protein microarrays are a high-throughput technology used increasingly in translational research, seeking to apply basic science findings to enhance human health. In addition to assessing protein levels, posttranslational modifications, and signaling pathways in patient samples, protein microarrays have aided in the identification of potential protein biomarkers of disease and infection. In this perspective, the different types of full-length protein microarrays that are used in translational research are reviewed. Specific studies employing these microarrays are presented to highlight their potential in finding solutions to real clinical problems. Finally, the criteria that should be considered when developing next-generation protein microarrays are provided. PMID:26749402

  12. Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities

    PubMed Central

    Berger, Michael F.; Philippakis, Anthony A.; Qureshi, Aaron M.; He, Fangxue S.; Estep, Preston W.; Bulyk, Martha L.

    2015-01-01

    Transcription factors (TFs) regulate the expression of genes involved in myriad cellular processes through sequence-specific interactions with DNA. In order to predict DNA regulatory elements and the TFs targeting them with greater accuracy, detailed knowledge of the binding preferences of TFs is needed. Protein binding microarray (PBM) technology permits rapid, high-throughput characterization of the in vitro DNA binding specificities of proteins1. Here, we present a novel, maximally compact, synthetic DNA sequence design that represents all possible DNA sequence variants of a given length k (i.e., all “k-mers”) on a single, universal microarray. We constructed such all k-mer microarrays covering all 10 base pair (bp) binding sites by converting high-density single-stranded oligonucleotide arrays to double-stranded DNA arrays. Using these microarrays, we comprehensively determined the binding specificities over a full range of affinities for five TFs of diverse structural classes from yeast, worm, mouse, and human. Importantly, the unbiased coverage of all k-mers permits an interrogation of binding site preferences, including nucleotide interdependencies, at unprecedented resolution. PMID:16998473

  13. PMD: A Resource for Archiving and Analyzing Protein Microarray data

    PubMed Central

    Xu, Zhaowei; Huang, Likun; Zhang, Hainan; Li, Yang; Guo, Shujuan; Wang, Nan; Wang, Shi-hua; Chen, Ziqing; Wang, Jingfang; Tao, Sheng-ce

    2016-01-01

    Protein microarray is a powerful technology for both basic research and clinical study. However, because there is no database specifically tailored for protein microarray, the majority of the valuable original protein microarray data is still not publically accessible. To address this issue, we constructed Protein Microarray Database (PMD), which is specifically designed for archiving and analyzing protein microarray data. In PMD, users can easily browse and search the entire database by experimental name, protein microarray type, and sample information. Additionally, PMD integrates several data analysis tools and provides an automated data analysis pipeline for users. With just one click, users can obtain a comprehensive analysis report for their protein microarray data. The report includes preliminary data analysis, such as data normalization, candidate identification, and an in-depth bioinformatics analysis of the candidates, which include functional annotation, pathway analysis, and protein-protein interaction network analysis. PMD is now freely available at www.proteinmicroarray.cn. PMID:26813635

  14. Integrated imaging instrument for self-calibrated fluorescence protein microarrays

    NASA Astrophysics Data System (ADS)

    Reddington, A. P.; Monroe, M. R.; Ünlü, M. S.

    2013-10-01

    Protein microarrays, or multiplexed and high-throughput assays, monitor multiple protein binding events to facilitate the understanding of disease progression and cell physiology. Fluorescence imaging is a popular method to detect proteins captured by immobilized probes with high sensitivity and specificity. Reliability of fluorescence assays depends on achieving minimal inter- and intra-assay probe immobilization variation, an ongoing challenge for protein microarrays. Therefore, it is desirable to establish a label-free method to quantify the probe density prior to target incubation to calibrate the fluorescence readout. Previously, a silicon oxide on silicon chip design was introduced to enhance the fluorescence signal and enable interferometric imaging to self-calibrate the signal with the immobilized probe density. In this paper, an integrated interferometric reflectance imaging sensor and wide-field fluorescence instrument is introduced for sensitive and calibrated microarray measurements. This platform is able to analyze a 2.5 mm × 3.4 mm area, or 200 spots (100 μm diameter with 200 μm pitch), in a single field-of-view.

  15. Prognostic Significance of CREB-Binding Protein and CD81 Expression in Primary High Grade Non-Muscle Invasive Bladder Cancer: Identification of Novel Biomarkers for Bladder Cancer Using Antibody Microarray.

    PubMed

    Lee, Myung-Shin; Kim, Joo Heon; Lee, Ji-Su; Yun, Seok Joong; Kim, Wun-Jae; Ahn, Hanjong; Park, Jinsung

    2015-01-01

    High-grade (HG) bladder cancers (BCs) are genetically unstable and have an unpredictable course. The identification of prognostic factors in HG non-muscle invasive BC (NMIBC) is crucial for improving patients' quality of life and preventing BC-specific mortality. Here, we used an antibody microarray (AbM) to identify novel candidate biomarkers in primary HG NMIBC and validated the prognostic significance of the candidate biomarkers. Three pairs of tissue samples from primary HG NMIBC and normal urothelium were analyzed using an AbM kit containing 656 antibodies, and differentially expressed proteins were identified. Among the 42 upregulated and 14 downregulated proteins with statistical significance in BC tissues, CREB-binding protein and CD81 were selected as representative upregulated and downregulated candidate biomarkers, respectively. We then validated the expression of these candidate biomarkers in primary human urothelial cells and BC cell lines by western blotting and immunofluorescence assays, and the results were consistent with the AbM expression profiles. Additionally, Kaplan-Meier survival using immunohistochemical data from an independent primary HG NMIBC cohort comprising 113 patients showed that expression of the 2 biomarkers was significantly associated with recurrence-free and progression-free survival. In multivariate analysis, the 2 biomarkers remained significant predictors for recurrence-free survival. Taken together, our findings suggest that expression of CREB-binding protein and CD81 in BC tissue specimens may have prognostic value in patients with primary HG NMIBC. PMID:25915404

  16. Studying cellular processes and detecting disease with protein microarrays

    SciTech Connect

    Zangar, Richard C.; Varnum, Susan M.; Bollinger, Nikki

    2005-10-31

    Protein microarrays are a rapidly developing analytic tool with diverse applications in biomedical research. These applications include profiling of disease markers or autoimmune responses, understanding molecular pathways, protein modifications and protein activities. One factor that is driving this expanding usage is the wide variety of experimental formats that protein microarrays can take. In this review, we provide a short, conceptual overview of the different approaches for protein microarray. We then examine some of the most significant applications of these microarrays to date, with an emphasis on how global protein analyses can be used to facilitate biomedical research.

  17. ProMAT: protein microarray analysis tool

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Varnum, Susan M.; Anderson, Kevin K.; Bollinger, Nikki; Zangar, Richard C.

    2006-04-04

    Summary: ProMAT is a software tool for statistically analyzing data from ELISA microarray experiments. The software estimates standard curves, sample protein concentrations and their uncertainties for multiple assays. ProMAT generates a set of comprehensive figures for assessing results and diagnosing process quality. The tool is available for Windows or Mac, and is distributed as open-source Java and R code. Availability: ProMAT is available at http://www.pnl.gov/statistics/ProMAT. ProMAT requires Java version 1.5.0 and R version 1.9.1 (or more recent versions) which are distributed with the tool.

  18. Fluorescent Protein Nanowire-Mediated Protein Microarrays for Multiplexed and Highly Sensitive Pathogen Detection.

    PubMed

    Men, Dong; Zhou, Juan; Li, Wei; Leng, Yan; Chen, Xinwen; Tao, Shengce; Zhang, Xian-En

    2016-07-13

    Protein microarrays are powerful tools for high-throughput and simultaneous detection of different target molecules in complex biological samples. However, the sensitivity of conventional fluorescence-labeling protein detection methods is limited by the availability of signal molecules for binding to the target molecule. Here, we built a multifunctional fluorescent protein nanowire (FNw) by harnessing self-assembly of yeast amyloid protein. The FNw integrated a large number of fluorescent molecules, thereby enhancing the fluorescent signal output in target detection. The FNw was then combined with protein microarray technology to detect proteins derived from two pathogens, including influenza virus (hemagglutinin 1, HA1) and human immunodeficiency virus (p24 and gp120). The resulting detection sensitivity achieved a 100-fold improvement over a commercially available detection reagent. PMID:27315221

  19. An ultralow background substrate for protein microarray technology.

    PubMed

    Feng, Hui; Zhang, Qingyang; Ma, Hongwei; Zheng, Bo

    2015-08-21

    We herein report an ultralow background substrate for protein microarrays. Conventional protein microarray substrates often suffer from non-specific protein adsorption and inhomogeneous spot morphology. Consequently, surface treatment and a suitable printing solution are required to improve the microarray performance. In the current work, we improved the situation by developing a new microarray substrate based on a fluorinated ethylene propylene (FEP) membrane. A polydopamine microspot array was fabricated on the FEP membrane, with proteins conjugated to the FEP surface through polydopamine. Uniform microspots were obtained on FEP without the application of a special printing solution. The modified FEP membrane demonstrated ultralow background signal and was applied in protein and peptide microarray analysis. PMID:26134063

  20. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  1. Protein microarrays based on polymer brushes prepared via surface-initiated atom transfer radical polymerization.

    PubMed

    Barbey, Raphael; Kauffmann, Ekkehard; Ehrat, Markus; Klok, Harm-Anton

    2010-12-13

    Polymer brushes represent an interesting platform for the development of high-capacity protein binding surfaces. Whereas the protein binding properties of polymer brushes have been investigated before, this manuscript evaluates the feasibility of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMA-co-PDEAEMA) (co)polymer brushes grown via surface-initiated atom transfer radical polymerization (SI-ATRP) as protein reactive substrates in a commercially available microarray system using tantalum-pentoxide-coated optical waveguide-based chips. The performance of the polymer-brush-based protein microarray chips is assessed using commercially available dodecylphosphate (DDP)-modified chips as the benchmark. In contrast to the 2D planar, DDP-coated chips, the polymer-brush-covered chips represent a 3D sampling volume. This was reflected in the results of protein immobilization studies, which indicated that the polymer-brush-based coatings had a higher protein binding capacity as compared to the reference substrates. The protein binding capacity of the polymer-brush-based coatings was found to increase with increasing brush thickness and could also be enhanced by copolymerization of 2-(diethylamino)ethyl methacrylate (DEAEMA), which catalyzes epoxide ring-opening of the glycidyl methacrylate (GMA) units. The performance of the polymer-brush-based microarray chips was evaluated in two proof-of-concept microarray experiments, which involved the detection of biotin-streptavidin binding as well as a model TNFα reverse assay. These experiments revealed that the use of polymer-brush-modified microarray chips resulted not only in the highest absolute fluorescence readouts, reflecting the 3D nature and enhanced sampling volume provided by the brush coating, but also in significantly enhanced signal-to-noise ratios. These characteristics make the proposed polymer brushes an attractive alternative to commercially available, 2D microarray

  2. The use of lectin microarray for assessing glycosylation of therapeutic proteins

    PubMed Central

    Zhang, Lei; Luo, Shen; Zhang, Baolin

    2016-01-01

    ABSTRACT Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins. PMID:26918373

  3. Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays.

    PubMed

    Serna, Sonia; Xolalpa, Wendy; Lang, Valérie; Aillet, Fabienne; England, Patrick; Reichardt, Niels; Rodriguez, Manuel S

    2016-08-01

    Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels. PMID:27410252

  4. Microintaglio Printing of In situ Synthesized Proteins Enables Rapid Printing of High-Density Protein Microarrays Directly from DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Biyani, Manish; Moriyasu, Junpei; Tanaka, Yoko; Sato, Shusuke; Ueno, Shingo; Ichiki, Takanori

    2013-08-01

    A simple and versatile approach to the simultaneous on-chip synthesis and printing of proteins has been studied for high-density protein microarray applications. The method used is based on the principle of intaglio printing using microengraved plates. Unlike conventional approaches that require multistep reactions for synthesizing proteins off the chip followed by printing using a robotic spotter, our approach demonstrates the following: (i) parallel and spotter-free printing of high-density protein microarrays directly from a type of DNA microarray and (ii) microcompartmentalization of cell-free coupled transcription/translation reaction and direct transferring of picoliter protein solution per spot to pattern microarrays of 25-100 µm features.

  5. Printing Proteins as Microarrays for High-Throughput Function Determination

    NASA Astrophysics Data System (ADS)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  6. Design of a combinatorial DNA microarray for protein-DNA interaction studies

    PubMed Central

    Mintseris, Julian; Eisen, Michael B

    2006-01-01

    Background Discovery of precise specificity of transcription factors is an important step on the way to understanding the complex mechanisms of gene regulation in eukaryotes. Recently, double-stranded protein-binding microarrays were developed as a potentially scalable approach to tackle transcription factor binding site identification. Results Here we present an algorithmic approach to experimental design of a microarray that allows for testing full specificity of a transcription factor binding to all possible DNA binding sites of a given length, with optimally efficient use of the array. This design is universal, works for any factor that binds a sequence motif and is not species-specific. Furthermore, simulation results show that data produced with the designed arrays is easier to analyze and would result in more precise identification of binding sites. Conclusion In this study, we present a design of a double stranded DNA microarray for protein-DNA interaction studies and show that our algorithm allows optimally efficient use of the arrays for this purpose. We believe such a design will prove useful for transcription factor binding site identification and other biological problems. PMID:17018151

  7. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  8. Label-free detection repeatability of protein microarrays by oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Dai, Jun; Li, Lin; Wang, JingYi; He, LiPing; Lu, HuiBin; Ruan, KangCheng; Jin, KuiJuan; Yang, GuoZhen

    2012-12-01

    We examine the repeatabilities of oblique-incidence reflectivity difference (OIRD) method for label-free detecting biological molecular interaction using protein microarrays. The experimental results show that the repeatabilities are the same in a given microarray or microarray-microarray and are consistent, indicating that OIRD is a promising label-free detection technique for biological microarrays.

  9. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  10. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  11. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    NASA Astrophysics Data System (ADS)

    Herbáth, Melinda; Papp, Krisztián; Balogh, Andrea; Matkó, János; Prechl, József

    2014-09-01

    Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

  12. Uncovering Quantitative Protein Interaction Networks for Mouse PDZ Domains using Protein Microarrays

    PubMed Central

    Stiffler, Michael A.; Grantcharova, Viara P.; Sevecka, Mark; MacBeath, Gavin

    2008-01-01

    One of the principle challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach – compatible with genome-wide investigations – that enables us to measure the equilibrium dissociation constant (KD) of recombinant PDZ domains for fluorescently-labeled peptides that represent physiologically-relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the KD for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (KD ≤ 10 μM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network. PMID:16637659

  13. Label and Label-Free Detection Techniques for Protein Microarrays

    PubMed Central

    Syahir, Amir; Usui, Kenji; Tomizaki, Kin-ya; Kajikawa, Kotaro; Mihara, Hisakazu

    2015-01-01

    Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano-biological events.

  14. Inkjet printing for the production of protein microarrays.

    PubMed

    McWilliam, Iain; Chong Kwan, Marisa; Hall, Duncan

    2011-01-01

    A significant proportion of protein microarray researchers would like the arrays they develop to become widely used research, screening, validation or diagnostic devices. For this to be achievable the arrays must be compatible with high-throughput techniques that allow manufacturing scale production. In order to simplify the transition from laboratory bench to market, Arrayjet have developed a range of inkjet microarray printers, which, at one end of the scale, are suitable for R&D and, at the other end, are capable of true high-throughput array output. To maintain scalability, all Arrayjet microarray printers utilise identical core technology comprising a JetSpyder™ liquid handling adaptor, which enables automated loading of an industry standard inkjet printhead compatible with non-contact on-the-fly printing. This chapter contains a detailed explanation of Arrayjet technology followed by a historical look at the development of inkjet technologies for protein microarray production. The method described subsequently is a simple example of an antibody array printed onto nitrocellulose-coated slides with specific detection with fluorescently labelled IgG. The method is linked to a notes section with advice on best practice and sources of useful information for protein microarray production using inkjet technology. PMID:21901611

  15. Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

    PubMed

    Fasolo, Joseph; Im, Hogune; Snyder, Michael P

    2015-01-01

    High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo. PMID:26274875

  16. Peptidoglycan microarray as a novel tool to explore protein-ligand recognition.

    PubMed

    Wang, Ning; Hirata, Akiyoshi; Nokihara, Kiyoshi; Fukase, Koichi; Fujimoto, Yukari

    2016-11-01

    Peptidoglycan is a giant bag-shaped molecule essential for bacterial cell shape and resistance to osmotic stresses. The activity of a large number of bacterial surface proteins involved in cell growth and division requires binding to this macromolecule. Recognition of peptidoglycan by immune effectors is also crucial for the establishment of the immune response against pathogens. The availability of pure and chemically defined peptidoglycan fragments is a major technical bottleneck that has precluded systematic studies of the mechanisms underpinning protein-mediated peptidoglycan recognition. Here, we report a microarray strategy suitable to carry out comprehensive studies to characterize proteins-peptidoglycan interactions. We describe a method to introduce a functional group on peptidoglycan fragments allowing their stable immobilization on amorphous carbon chip plates to minimize nonspecific binding. Such peptidoglycan microarrays were used with a model peptidoglycan binding protein-the human peptidoglycan recognition protein-S (hPGRP-S). We propose that this strategy could be implemented to carry out high-throughput analyses to study peptidoglycan binding proteins. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 422-429, 2016. PMID:26773558

  17. Design of a combinatorial dna microarray for protein-dnainteraction studies

    SciTech Connect

    Mintseris, Julian; Eisen, Michael B.

    2006-07-07

    Background: Discovery of precise specificity oftranscription factors is an important step on the way to understandingthe complex mechanisms of gene regulation in eukaryotes. Recently,doublestranded protein-binding microarrays were developed as apotentially scalable approach to tackle transcription factor binding siteidentification. Results: Here we present an algorithmic approach toexperimental design of a microarray that allows for testing fullspecificity of a transcription factor binding to all possible DNA bindingsites of a given length, with optimally efficient use of the array. Thisdesign is universal, works for any factor that binds a sequence motif andis not species-specific. Furthermore, simulation results show that dataproduced with the designed arrays is easier to analyze and would resultin more precise identification of binding sites. Conclusion: In thisstudy, we present a design of a double stranded DNA microarray forprotein-DNA interaction studies and show that our algorithm allowsoptimally efficient use of the arrays for this purpose. We believe such adesign will prove useful for transcription factor binding siteidentification and other biological problems.

  18. Nanotechnology in the Fabrication of Protein Microarrays.

    PubMed

    Fuentes, Manuel; Díez, Paula; Casado-Vela, Juan

    2016-01-01

    Protein biochips are the heart of many medical and bioanalytical applications. Increasing interest of protein biochip fabrication has been focused on surface activation and subsequent functionalization strategies for the immobilization of these molecules. PMID:26614077

  19. NAPPA as a Real New Method for Protein Microarray Generation

    PubMed Central

    Díez, Paula; González-González, María; Lourido, Lucía; Dégano, Rosa M.; Ibarrola, Nieves; Casado-Vela, Juan; LaBaer, Joshua; Fuentes, Manuel

    2015-01-01

    Nucleic Acid Programmable Protein Arrays (NAPPA) have emerged as a powerful and innovative technology for the screening of biomarkers and the study of protein-protein interactions, among others possible applications. The principal advantages are the high specificity and sensitivity that this platform offers. Moreover, compared to conventional protein microarrays, NAPPA technology avoids the necessity of protein purification, which is expensive and time-consuming, by substituting expression in situ with an in vitro transcription/translation kit. In summary, NAPPA arrays have been broadly employed in different studies improving knowledge about diseases and responses to treatments. Here, we review the principal advances and applications performed using this platform during the last years.

  20. Method for printing functional protein microarrays

    NASA Technical Reports Server (NTRS)

    Delehanty, James B.; Ligler, Frances S.

    2003-01-01

    Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.

  1. Probing small-molecule microarrays with tagged proteins in cell lysates.

    PubMed

    Pop, Marius S; Wassaf, Dina; Koehler, Angela N

    2014-01-01

    The technique of small-molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal-to-noise ratios (SNRs). SMM screening is a high-throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding-based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment. PMID:25445177

  2. Discovering cancer biomarkers from clinical samples by protein microarrays.

    PubMed

    Hu, Bin; Niu, Xin; Cheng, Li; Yang, Li-Na; Li, Qing; Wang, Yang; Tao, Sheng-Ce; Zhou, Shu-Min

    2015-02-01

    Cancer biomarkers are of potential use in early cancer diagnosis, anticancer therapy development, and monitoring the responses to treatments. Protein-based cancer biomarkers are major forms in use, as they are much easier to be monitored in body fluids or tissues. For cancer biomarker discovery, high-throughput techniques such as protein microarrays hold great promises, because they are capable of global unbiased monitoring but with a miniaturized format. In doing so, novel and cancer type specific biomarkers can be systematically discovered at an affordable cost. In this review, we give a relatively complete picture on protein microarrays applied to clinical samples for cancer biomarker discovery, and conclude this review with the future perspectives. PMID:25523829

  3. A microarray immunoassay for simultaneous detection of proteins and bacteria

    NASA Technical Reports Server (NTRS)

    Delehanty, James B.; Ligler, Frances S.

    2002-01-01

    We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).

  4. A versatile protein microarray platform enabling antibody profiling against denatured proteins

    PubMed Central

    Wang, Jie; Barker, Kristi; Steel, Jason; Park, Jin; Saul, Justin; Festa, Fernanda; Wallstrom, Garrick; Yu, Xiaobo; Bian, Xiaofang; Anderson, Karen S; Figueroa, Jonine D; LaBaer, Joshua; Qiu, Ji

    2014-01-01

    Purpose We aim to develop a protein microarray platform capable of presenting both natural and denatured forms of proteins for antibody biomarker discovery. We will further optimize plasma screening protocols to improve detection. Experimental design We developed a new covalent capture protein microarray chemistry using HaloTag fusion proteins and ligand. To enhance protein yield, we used HeLa cell lysate as an in vitro transcription translation system (IVTT). E. coli lysates were added to the plasma blocking buffer to reduce non-specific background. These protein microarrays were probed with plasma samples and autoantibody responses were quantified and compared with or without denaturing buffer treatment. Results We demonstrated that protein microarrays using the covalent attachment chemistry endured denaturing conditions. Blocking with E. coli lysates greatly reduced the background signals and expression with IVTT based on HeLa cell lysates significantly improved the antibody signals on protein microarrays probed with plasma samples. Plasma samples probed on denatured protein arrays produced autoantibody profiles distinct from those probed on natively displayed proteins. Conclusions and clinical relevance This versatile protein microarray platform allows the display of both natural and denatured proteins, offers a new dimension to search for disease-specific antibodies, broadens the repertoire of potential biomarkers, and will potentially yield clinical diagnostics with greater performance. PMID:23027520

  5. Reverse phase protein microarrays advance to use in clinical trials.

    PubMed

    Mueller, Claudius; Liotta, Lance A; Espina, Virginia

    2010-12-01

    Individualizing cancer therapy for molecular targeted inhibitors requires a new class of molecular profiling technology that can map the functional state of the cancer cell signal pathways containing the drug targets. Reverse phase protein microarrays (RPMA) are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and non-phosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate tissue samples, cells, serum, or body fluids. RPMA were previously a research tool; now this technology has graduated to use in research clinical trials with clinical grade sensitivity and precision. In this review we describe the application of RPMA for multiplexed signal pathway analysis in therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets, and conclude with a summary of the technical aspects of RPMA construction and analysis. PMID:20974554

  6. RCA combined nanoparticle-based optical detection technique for protein microarray: a novel approach.

    PubMed

    Hsu, Hsin-Yun; Huang, Yi-You

    2004-07-30

    Developing a readily available biosensor with excellent performances is the main focus of many research groups. Recently, major breakthroughs in miniaturization of molecular analysis have produced DNA and protein microarrays. The aim of our group is to develop a sensitive technique for analyzing signals on protein microarray by applying the surface plasmon resonance (SPR) method. This new detection technique for specific molecular binding utilizes rolling circles amplification (RCA) post-signal processing method [Nat. Genet. 19 (1998) 225-232] and optical visualization by nanogold particle-labeled molecules on a micro-structured chip surface. By covalent bonding of the RCA primer to the detection antibody guarantees that the linkage between the analyte and the amplified RCA product is maintained during the assay. Experimental results show that RCA has significantly enhanced sensitivity compared to conventional methods. This combination of an easily detectable signal with chip technology should have the potential to become a successful commercial application. PMID:15142584

  7. Tracking Humoral Responses Using Self Assembling Protein Microarrays

    PubMed Central

    Ramachandran, Niroshan; Anderson, Karen S.; Raphael, Jacob v.; Hainsworth, Eugenie; Sibani, Sahar; Montor, Wagner R.; Pacek, Marcin; Wong, Jessica; Eljanne, Mariam; Sanda, Martin G.; Hu, Yanhui; Logvinenko, Tanya; LaBaer, Joshua

    2011-01-01

    The humoral immune response is a highly specific and adaptive sensor for changes in the body's protein milieu, which responds to novel structures of both foreign and self antigens. Although immunoglobulins represent a major component of human serum and are vital to survival, little is known about the response specificity and determinants that govern the human immunome. Historically, antigen-specific humoral immunity has been investigated using individually-produced and purified target proteins, a labor-intensive process that has limited the number of antigens that have been studied. Here, we present the development of methods for applying self-assembling protein microarrays and a related method for producing 96-well formatted macroarrays for monitoring the humoral response at the proteome scale. Using plasmids encoding full-length cDNAs for over 850 human proteins and 1700 pathogen proteins, we demonstrate that these microarrays are highly sensitive, specific, reproducible, and can simultaneously measure immunity to thousands of proteins without a priori protein purification. Using this approach, we demonstrate the detection of humoral immunity to known and novel self-antigens, cancer antigens, autoimmune antigens, as well as pathogen-derived antigens. This represents a powerful and versatile tool for monitoring the immunome in health and disease. PMID:21136799

  8. Quantum Dots-based Reverse Phase Protein Microarray

    SciTech Connect

    Shingyoji, Masato; Gerion, Daniele; Pinkel, Dan; Gray, Joe W.; Chen, Fanqing

    2005-07-15

    CdSe nanocrystals, also called quantum dots (Qdots) are a novel class of fluorophores, which have a diameter of a few nanometers and possess high quantum yield, tunable emission wavelength and photostability. They are an attractive alternative to conventional fluorescent dyes. Quantum dots can be silanized to be soluble in aqueous solution under biological conditions, and thus be used in bio-detection. In this study, we established a novel Qdot-based technology platform that can perform accurate and reproducible quantification of protein concentration in a crude cell lysate background. Protein lysates have been spiked with a target protein, and a dilution series of the cell lysate with a dynamic range of three orders of magnitude has been used for this proof-of-concept study. The dilution series has been spotted in microarray format, and protein detection has been achieved with a sensitivity that is at least comparable to standard commercial assays, which are based on horseradish peroxidase (HRP) catalyzed diaminobenzidine (DAB) chromogenesis. The data obtained through the Qdot method has shown a close linear correlation between relative fluorescence unit and relative protein concentration. The Qdot results are in almost complete agreement with data we obtained with the well-established HRP-DAB colorimetric array (R{sup 2} = 0.986). This suggests that Qdots can be used for protein quantification in microarray format, using the platform presented here.

  9. Cold Spots in Protein Binding.

    PubMed

    Shirian, Jason; Sharabi, Oz; Shifman, Julia M

    2016-09-01

    Understanding the energetics and architecture of protein-binding interfaces is important for basic research and could potentially facilitate the design of novel binding domains for biotechnological applications. It is well accepted that a few key residues at binding interfaces (binding hot spots) are responsible for contributing most to the free energy of binding. In this opinion article, we introduce a new concept of 'binding cold spots', or interface positions occupied by suboptimal amino acids. Such positions exhibit a potential for affinity enhancement through various mutations. We give several examples of cold spots from different protein-engineering studies and argue that identification of such positions is crucial for studies of protein evolution and protein design. PMID:27477052

  10. Multiplexed electrospray deposition for protein microarray with micromachined silicon device

    NASA Astrophysics Data System (ADS)

    Bhatnagar, Parijat

    2007-07-01

    Multiplexed electrospray deposition device capable of delivering picoliter volumes made by silicon micromachining technology has been developed as a deposition tool for making protein microarrays in a noncontact mode. Upon application of potential difference in the range of 7-9kV, biomolecules dissolved in suitable buffer with nonionic surfactant and loaded on the electrospray tips were dispensed on the substrate with microfabricated hydrogel features (1-10μm) in cone-jet mode. Schiff base chemistry followed by reductive amination was utilized for covalent immobilization.

  11. Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling.

    PubMed

    Sjöberg, Ronald; Mattsson, Cecilia; Andersson, Eni; Hellström, Cecilia; Uhlen, Mathias; Schwenk, Jochen M; Ayoglu, Burcu; Nilsson, Peter

    2016-09-25

    High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions. PMID:26417875

  12. High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

    PubMed Central

    Moller, Isabel; Marcus, Susan E.; Haeger, Ash; Verhertbruggen, Yves; Verhoef, Rene; Schols, Henk; Ulvskov, Peter; Mikkelsen, Jørn Dalgaard; Knox, J. Paul

    2007-01-01

    Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials. PMID:17629746

  13. Human protein atlas and the use of microarray technologies.

    PubMed

    Hober, S; Uhlén, M

    2008-02-01

    Currently one of the most challenging tasks in biological and medical research is to explore and understand the function of all proteins encoded by the genome of an organism. A systematic approach based on the genome sequences is feasible because the full genome of many organisms presently is available and many more are underway. For the production of expression atlases different strategies are used. Early attempts to acquire information about protein expression levels have focused on the analysis of mRNA levels within different tissues and cell types. Recently, novel strategies to focus directly on protein levels have been developed. To assess global protein expression in a systematic and high-throughput manner, methods based on design of specific affinity ligands to recognize the proteins have been presented. By subsequently using these affinity molecules for detection of the corresponding proteins in a wide range of platforms, important information can be gained. This article focuses on strategies to profile protein levels and in particular the human protein atlas initiative and the use of microarray technologies. PMID:18187316

  14. Binding Efficiency of Protein-Protein Complexes

    PubMed Central

    Day, Eric S.; Cote, Shaun M.; Whitty, Adrian

    2012-01-01

    We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPI), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for the large receptor TNFR1 binding to its ligands TNFα (KD = 1.4 ± 0.4 nM) and lymphotoxin-α (KD = 50 ± 10 nM), and also for the small receptor Fn14 binding to TWEAK (KD = 70 ± 10 nM). We additionally assembled data for all other TNF/TNFR family complexes for which reliable single site binding affinities have been reported. We used these values to calculate the binding efficiency – defined as binding energy per Å2 of surface area buried at the contact interface – for the nine of these complexes for which co-crystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinity values. The results show that the most efficient PPI complexes generate ~20 cal.mol−1/Å2 of binding energy. A minimum contact area of ~500 Å2 is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of co-localizing two proteins from 1 M solution. The most compact and efficient TNF/TNFR complex was BAFF/BR3, which achieved ~80% of the maximum achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method to predict how large a natural or engineered contact interface must be to achieve a given level of binding affinity. PMID:23088250

  15. Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples*

    PubMed Central

    Pla-Roca, M.; Leulmi, R. F.; Tourekhanova, S.; Bergeron, S.; Laforte, V.; Moreau, E.; Gosline, S. J. C.; Bertos, N.; Hallett, M.; Park, M.; Juncker, D.

    2012-01-01

    DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable. PMID:22171321

  16. When is protein binding important?

    PubMed

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. PMID:23650013

  17. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    PubMed

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity. PMID:26643897

  18. [Binding capability of lidamycin apoprotein to human breast cancer detected by tissue microarrays].

    PubMed

    Cai, Lin; Gao, Rui-Juan; Guo, Xiao-Zhong; Li, Yi; Zhen, Yong-Su

    2010-05-01

    This study is to investigate the binding capability of lidamycin apoprotein (LDP), an enediyne-associated apoprotein of the chromoprotein antitumor antibiotic family, to human breast cancer and normal tissues, the correlation of LDP binding capability to human breast cancer tissues and the expression of tumor therapeutic targets such as VEGF and HER2. In this study, the binding capability of LDP to human breast cancer tissues was detected with tissue microarray. The correlation study of LDP binding capability to human breast tumor tissues and relevant therapeutic targets was performed on breast cancer tissue microarrays. Immunocytochemical examination was used to detect the binding capability of LDP to human breast carcinoma MCF-7 cells. As a result, tissue microarray showed that LDP staining of 73.2% (30/41) of breast cancer tissues was positive, whereas that of 48.3% (15/31) of the adjacent normal breast specimens was positive. The difference between the tumor and normal samples was significant (Chi2 = 4.63, P < 0.05). LDP immunoreactivity in breast cancer correlated significantly with the overexpression of VEGF and HER2 (P < 0.001 and < 0.01, r = 0.389 and 0.287, respectively). Determined with confocal immunofluorescent analysis, LDP showed the binding capability to mammary carcinoma MCF-7 cells. It is demonstrated that LDP can bind to human breast cancer tissues and there is significant difference between the breast cancer tissues and the corresponding normal tissues. Notably, the binding reactivity shows positive correlation with the expression of VEGF and HER2 in breast carcinoma tissues. The results imply that LDP may have a potential use as targeting drug carrier in the research and development of new anticancer therapeutics. This study may provide reference for drug combination of LDM and other therapeutic agents. PMID:20931759

  19. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

    PubMed Central

    Yu, Xiaobo; LaBaer, Joshua

    2015-01-01

    Summary AMPylation (adenylylation) has been recognized as an important post translational modification employed by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes and is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method to identify new substrates using protein microarrays, which can significantly expand the list of potential substrates. Here, we describe procedures to detect AMPylated and auto-AMPylated proteins in a sensitive, high throughput, and non-radioactive manner. The approach employs high-density protein microarrays fabricated using NAPPA (Nucleic Acid Programmable Protein Arrays) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide–alkyne cycloaddition. The assay can be accomplished within 11 hours. PMID:25881200

  20. Protein Microarrays-Based Strategies for Life Detection in Astrobiology

    NASA Astrophysics Data System (ADS)

    Parro, Víctor; Rivas, Luis A.; Gómez-Elvira, Javier

    2008-03-01

    The detection of organic molecules of unambiguous biological origin is fundamental for the confirmation of present or past life. Planetary exploration requires the development of miniaturized apparatus for in situ life detection. Analytical techniques based on mass spectrometry have been traditionally used in space science. Following the Viking landers, gas chromatography-mass spectrometry (GC-MS) for organic detection has gained general acceptance and has been used successfully in the Cassini-Huygens mission to Titan. Microfluidics allows the development of miniaturized capillary electrophoresis devices for the detection of important molecules for life, like amino acids or nucleobases. Recently, a new approach is gaining acceptance in the space science community: the application of the well-known, highly specific, antibody-antigen affinity interaction for the detection and identification of organics and biochemical compounds. Antibodies can specifically bind a plethora of structurally different compounds of a broad range of molecular sizes, from amino acids level to whole cells. Antibody microarray technology allows us to look for the presence of thousands of different compounds in a single assay and in just one square centimeter. Herein, we discuss several important issues—most of which are common with other instruments dealing with life signature detection in the solar system—that must be addressed in order to use antibody microarrays for life detection and planetary exploration. These issues include (1) preservation of biomarkers, (2) the extraction techniques for biomarkers, (3) terrestrial analogues, (4) the antibody stability under space environments, (5) the selection of unequivocal biomarkers for the antibody production, or (6) the instrument design and implementation.

  1. Protein Microarrays-Based Strategies for Life Detection in Astrobiology

    NASA Astrophysics Data System (ADS)

    Parro, Víctor; Rivas, Luis A.; Gómez-Elvira, Javier

    The detection of organic molecules of unambiguous biological origin is fundamental for the confirmation of present or past life. Planetary exploration requires the development of miniaturized apparatus for in situ life detection. Analytical techniques based on mass spectrometry have been traditionally used in space science. Following the Viking landers, gas chromatography-mass spectrometry (GC-MS) for organic detection has gained general acceptance and has been used successfully in the Cassini-Huygens mission to Titan. Microfluidics allows the development of miniaturized capillary electrophoresis devices for the detection of important molecules for life, like amino acids or nucleobases. Recently, a new approach is gaining acceptance in the space science community: the application of the well-known, highly specific, antibody-antigen affinity interaction for the detection and identification of organics and biochemical compounds. Antibodies can specifically bind a plethora of structurally different compounds of a broad range of molecular sizes, from amino acids level to whole cells. Antibody microarray technology allows us to look for the presence of thousands of different compounds in a single assay and in just one square centimeter. Herein, we discuss several important issues—most of which are common with other instruments dealing with life signature detection in the solar system—that must be addressed in order to use antibody microarrays for life detection and planetary exploration. These issues include (1) preservation of biomarkers, (2) the extraction techniques for biomarkers, (3) terrestrial analogues, (4) the antibody stability under space environments, (5) the selection of unequivocal biomarkers for the antibody production, or (6) the instrument design and implementation.

  2. Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

    PubMed Central

    Stieber, Bettina; Monecke, Stefan; Müller, Elke; Büchler, Joseph; Ehricht, Ralf

    2015-01-01

    Background S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins. Methods In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays. Results 110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate. Conclusions The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers. PMID:26624622

  3. Protein microarrays imaging using oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Lu, Heng; Zhang, Hongyan; Li, Wei; Zhou, Yueliang; Lu, Huibin; Jin, Kuijuan; Yang, Guozhen; Ruan, Kangcheng

    2007-11-01

    In this study Oblique-Incidence Reflectivity Difference (OIRD) technique, a recent developed label-free detection, is applied to image biomolecular microarrays. Compared to the currently widely used fluorescence-dependent optical microscopy, OIRD technique not only images the morphology of protein bio-arrays in the absence of extrinsic labeling molecules but also monitors the changes in the optical properties of biochips in high-throughput fashion. Additionally, such a technique complements other label-free detections including Surface Plasmon Resonance (SPR) and Mass Spectrometry (MS) by offering the opportunity to detect biochemical activities without the special requirements on the substrate or the specific matrix medium. It is shown in this article that the surface topography can be reflected by OIRD method. Besides, the differences among printing concentrations and various proteins are able to be identified as well. Incidentally, the OIRD images appear to be useful aids to distinguish the variations resulting from antibody-antigen capture. Both the imaginary and real parts of OIRD images we presented provide more information than the single imaginary one especially. On the basis of the scattering mechanism, the absorptive properties of protein molecules are analyzed through the real part of OIRD signal. Accordingly, OIRD technique shows its unique potential in detection of biochemical processes.

  4. The prion protein binds thiamine.

    PubMed

    Perez-Pineiro, Rolando; Bjorndahl, Trent C; Berjanskii, Mark V; Hau, David; Li, Li; Huang, Alan; Lee, Rose; Gibbs, Ebrima; Ladner, Carol; Dong, Ying Wei; Abera, Ashenafi; Cashman, Neil R; Wishart, David S

    2011-11-01

    Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ~60 μM. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N-terminal region in the presence and absence of copper, were examined. This indicates that the N-terminus has no influence on the protein's ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine's interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer-aided docking and molecular dynamics, were used to model the thiamine-binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction. PMID:21848803

  5. Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches

    PubMed Central

    Rogers, Claude J.; Clark, Peter M.; Tully, Sarah E.; Abrol, Ravinder; Garcia, K. Christopher; Goddard, William A.; Hsieh-Wilson, Linda C.

    2011-01-01

    Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan–protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan–protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan–protein–protein interactions, as well as a molecular-level understanding of those complexes. PMID:21628576

  6. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  7. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  8. Identifying protein interactions with metal-modified DNA using microarray technology.

    PubMed

    Stansfield, Hope E; Kulczewski, Bethany P; Lybrand, Kyle E; Jamieson, Elizabeth R

    2009-02-01

    Protein microarrays have been used extensively to identify protein-protein interactions; however, this technology has not been widely applied to protein-DNA interactions. In particular, this work demonstrates the utility of this technique for rapidly identifying interactions of proteins with metal-modified DNA. Protein macroarray experiments were carried out with high mobility group protein 1 (HMG-1) and cisplatin- and chromium-modified 50-mer oligonucleotides to demonstrate "proof of principle." Commercially available protein microarrays containing many different classes of human proteins were then employed to search for additional interactions with cisplatin-modified DNA. The results of the microarray experiments confirmed some known interactions and, more importantly, identified many novel protein interactions, demonstrating the utility of this method as a rapid, high-throughput technique to discover proteins that interact with metal-modified DNA. PMID:18936984

  9. Anti-heat shock protein autoantibody profiling in breast cancer using customized protein microarray.

    PubMed

    Shi, Liu; Gehin, Thomas; Chevolot, Yann; Souteyrand, Eliane; Mangé, Alain; Solassol, Jérôme; Laurenceau, Emmanuelle

    2016-02-01

    Heat shock proteins (HSPs) are over-expressed in a wide range of human cancers. It results in the stimulation of the immune system and consequently in elevated concentration of anti-HSP autoantibodies. Elevated anti-HSP autoantibodies were found in breast cancer patients, and they are associated with tumor metastasis. Therefore, screening these autoantibodies could be of diagnostic and prognostic values. Protein microarrays have already demonstrated their great potential as a diagnostic tool. However, protein diversity requires optimization of the microarray fabrication to achieve high sensitivity and specificity. In this study, seven HSPs were immobilized on six different surface chemistries. After evaluation and optimization with purified antibodies of the six surface chemistries, two surfaces were selected to detect anti-HSP autoantibodies in breast cancer sera. Multiplex detection of anti-HSP autoantibodies allowed discrimination of breast cancer patients (50) from healthy controls (26) with a sensitivity of 86% and a specificity of 100%. PMID:26715250

  10. High-throughput prediction of protein antigenicity using protein microarray data

    PubMed Central

    Magnan, Christophe N.; Zeller, Michael; Kayala, Matthew A.; Vigil, Adam; Randall, Arlo; Felgner, Philip L.; Baldi, Pierre

    2010-01-01

    Motivation: Discovery of novel protective antigens is fundamental to the development of vaccines for existing and emerging pathogens. Most computational methods for predicting protein antigenicity rely directly on homology with previously characterized protective antigens; however, homology-based methods will fail to discover truly novel protective antigens. Thus, there is a significant need for homology-free methods capable of screening entire proteomes for the antigens most likely to generate a protective humoral immune response. Results: Here we begin by curating two types of positive data: (i) antigens that elicit a strong antibody response in protected individuals but not in unprotected individuals, using human immunoglobulin reactivity data obtained from protein microarray analyses; and (ii) known protective antigens from the literature. The resulting datasets are used to train a sequence-based prediction model, ANTIGENpro, to predict the likelihood that a protein is a protective antigen. ANTIGENpro correctly classifies 82% of the known protective antigens when trained using only the protein microarray datasets. The accuracy on the combined dataset is estimated at 76% by cross-validation experiments. Finally, ANTIGENpro performs well when evaluated on an external pathogen proteome for which protein microarray data were obtained after the initial development of ANTIGENpro. Availability: ANTIGENpro is integrated in the SCRATCH suite of predictors available at http://scratch.proteomics.ics.uci.edu. Contact: pfbaldi@ics.uci.edu PMID:20934990

  11. Dye-Doped Silica Nanoparticle Labels/Protein Microarray for Detection of Protein Biomarkers

    SciTech Connect

    Wu, Hong; Huo, Qisheng; Varnum, Susan M.; Liu, Guodong; Wang, Jun; Nie, Zimin; Liu, Jun; Lin, Yuehe

    2008-10-20

    Biomarkers serve as indicators of biological and pathological processes, or physiological and pharmacological responses to a drug treatment. Interleukin-6 (IL-6), a biomarker with its important biological and pathological functions, has been studied for decades. Conventional fluorescence immunoassay has been widely used for analysis of biomakers like IL-6. However, single fluorophore labeling shows its limitations of low intensity and poor stability. We report a dye-encapsulated silica nanoparticle as a label, with the advantages of high fluorescence intensity, photostability, and biocompatibility, in conjunction with microarray technology for sensitive immunoassay of IL-6 on a microarray format. The tris (2,2’-bipyridyl)ruthenium (II)chloride hexahydrate (Rubpy) dye incorporated into silica nanoparticles using a simple one-step microemulsion synthesis step. The nanoparticles are uniform in size with a diameter of 50 nm. The microarray fluorescent immunoassay approach based on dye-doped silica nanoparticle labels has high sensitivity for practical applications with a limit of detection for IL-6 down to 0.1 ng mL-1. The calibration curve is linear over the range from 0.1 ng mL-1 to 10 ng mL-1. Furthermore, results illustrated that the assay is highly specific for IL-6 in the presence of range of cytokines or proteins. The RuDS dye-labeled nanoparticles in connection with protein microarrays show the promise for clinical diagnosis of biomarkers.

  12. Detection of antibodies against avian influenza virus by protein microarray using nucleoprotein expressed in insect cells

    PubMed Central

    ZHAO, Yuhui; WANG, Xiurong; CHEN, Pucheng; ZENG, Xianying; BAO, Hongmei; WANG, Yunhe; XU, Xiaolong; JIANG, Yongping; CHEN, Hualan; LI, Guangxing

    2014-01-01

    Avian influenza (AI) is an infectious disease caused by avian influenza viruses (AIVs) which belong to the influenza virus A group. AI causes tremendous economic losses in poultry industry and pose great threatens to human health. Active serologic surveillance is necessary to prevent and control the spread of AI. In this study, a protein microarray using nucleoprotein (NP) of H5N1 AIV expressed in insect cells was developed to detect antibodies against AIV NP protein. The protein microarray was used to test Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), AIV positive and negative sera. The results indicated that the protein microarray could hybridize specifically with antibodies against AIV with strong signals and without cross-hybridization. Moreover, 76 field serum samples were detected by microarray, enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition test (HI). The positive rate was 92.1% (70/76), 93.4% (71/76) and 89.4% (68/76) by protein microarray, ELISA and HI test, respectively. Compared with ELISA, the microarray showed 100% (20/20) agreement ratio in chicken and 98.2% (55/56) in ornamental bird. In conclusion, this method provides an alternative serological diagnosis for influenza antibody screening and will provide a basis for the development of protein microarrays that can be used to respectively detect antibodies of different AIV subtypes and other pathogens. PMID:25650059

  13. Detection of antibodies against avian influenza virus by protein microarray using nucleoprotein expressed in insect cells.

    PubMed

    Zhao, Yuhui; Wang, Xiurong; Chen, Pucheng; Zeng, Xianying; Bao, Hongmei; Wang, Yunhe; Xu, Xiaolong; Jiang, Yongping; Chen, Hualan; Li, Guangxing

    2015-04-01

    Avian influenza (AI) is an infectious disease caused by avian influenza viruses (AIVs) which belong to the influenza virus A group. AI causes tremendous economic losses in poultry industry and pose great threatens to human health. Active serologic surveillance is necessary to prevent and control the spread of AI. In this study, a protein microarray using nucleoprotein (NP) of H5N1 AIV expressed in insect cells was developed to detect antibodies against AIV NP protein. The protein microarray was used to test Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), AIV positive and negative sera. The results indicated that the protein microarray could hybridize specifically with antibodies against AIV with strong signals and without cross-hybridization. Moreover, 76 field serum samples were detected by microarray, enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition test (HI). The positive rate was 92.1% (70/76), 93.4% (71/76) and 89.4% (68/76) by protein microarray, ELISA and HI test, respectively. Compared with ELISA, the microarray showed 100% (20/20) agreement ratio in chicken and 98.2% (55/56) in ornamental bird. In conclusion, this method provides an alternative serological diagnosis for influenza antibody screening and will provide a basis for the development of protein microarrays that can be used to respectively detect antibodies of different AIV subtypes and other pathogens. PMID:25650059

  14. Protein Microarrays with Novel Microfluidic Methods: Current Advances

    PubMed Central

    Dixit, Chandra K.; Aguirre, Gerson R.

    2014-01-01

    Microfluidic-based micromosaic technology has allowed the pattering of recognition elements in restricted micrometer scale areas with high precision. This controlled patterning enabled the development of highly multiplexed arrays multiple analyte detection. This arraying technology was first introduced in the beginning of 2001 and holds tremendous potential to revolutionize microarray development and analyte detection. Later, several microfluidic methods were developed for microarray application. In this review we discuss these novel methods and approaches which leverage the property of microfluidic technologies to significantly improve various physical aspects of microarray technology, such as enhanced imprinting homogeneity, stability of the immobilized biomolecules, decreasing assay times, and reduction of the costs and of the bulky instrumentation.

  15. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

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

    PubMed

    Franco-Zorrilla, José M; Solano, Roberto

    2014-01-01

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

  17. A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

    PubMed Central

    Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

    2012-01-01

    We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

  18. Detection of protein microarrays by oblique-incidence reflectivity difference technique

    NASA Astrophysics Data System (ADS)

    Wen, Juan; Lu, Heng; Wang, Xu; Yuan, Kun; Lü, Huibin; Zhou, Yueliang; Yin, Kuijuan; Yang, Guozhen; Li, Wei; Ruan, Kangcheng

    2010-02-01

    Biological microarrays with different proteins and different protein concentrations are detected without external labeling by an oblique-incidence reflectivity difference (OIRD) technique. The initial experiment results reveal that the intensities of OIRD signals can distinguish the different proteins and concentrations of protein. The OIRD technique promises feasible applications to life sciences for label-free and high-throughput detection.

  19. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h. PMID:25881200

  20. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  1. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, Gisela K.

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  2. Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

    PubMed Central

    Lu, Chen; Wonsidler, Joshua L.; Li, Jianwei; Du, Yanming; Block, Timothy; Haab, Brian; Chen, Songming

    2012-01-01

    In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed. Introduction Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies

  3. Baculovirus display for discovery of low-affinity extracellular receptor-ligand interactions using protein microarrays.

    PubMed

    Tom, Irene; Estevez, Alberto; Bowman, Krista; Gonzalez, Lino C

    2015-06-15

    When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors. PMID:25797350

  4. Small Molecule Microarrays Enable the Identification of a Selective, Quadruplex-Binding Inhibitor of MYC Expression

    PubMed Central

    2015-01-01

    The transcription factor MYC plays a pivotal role in cancer initiation, progression, and maintenance. However, it has proven difficult to develop small molecule inhibitors of MYC. One attractive route to pharmacological inhibition of MYC has been the prevention of its expression through small molecule-mediated stabilization of the G-quadruplex (G4) present in its promoter. Although molecules that bind globally to quadruplex DNA and influence gene expression are well-known, the identification of new chemical scaffolds that selectively modulate G4-driven genes remains a challenge. Here, we report an approach for the identification of G4-binding small molecules using small molecule microarrays (SMMs). We use the SMM screening platform to identify a novel G4-binding small molecule that inhibits MYC expression in cell models, with minimal impact on the expression of other G4-associated genes. Surface plasmon resonance (SPR) and thermal melt assays demonstrated that this molecule binds reversibly to the MYC G4 with single digit micromolar affinity, and with weaker or no measurable binding to other G4s. Biochemical and cell-based assays demonstrated that the compound effectively silenced MYC transcription and translation via a G4-dependent mechanism of action. The compound induced G1 arrest and was selectively toxic to MYC-driven cancer cell lines containing the G4 in the promoter but had minimal effects in peripheral blood mononucleocytes or a cell line lacking the G4 in its MYC promoter. As a measure of selectivity, gene expression analysis and qPCR experiments demonstrated that MYC and several MYC target genes were downregulated upon treatment with this compound, while the expression of several other G4-driven genes was not affected. In addition to providing a novel chemical scaffold that modulates MYC expression through G4 binding, this work suggests that the SMM screening approach may be broadly useful as an approach for the identification of new G4-binding small

  5. Identification of Biologically Active, HIV TAR RNA-Binding Small Molecules Using Small Molecule Microarrays

    PubMed Central

    2015-01-01

    Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to find RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. Here we demonstrate a strategy to screen a large unbiased library of druglike small molecules in a microarray format against an RNA target. This approach has enabled the identification of a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. Thienopyridine 4 binds to and stabilizes the TAR hairpin with a Kd of 2.4 μM. Structure–activity relationships demonstrate that this compound achieves activity through hydrophobic and aromatic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5′ untranslated region (UTR) of the HIV-1 genome to determine global structural changes in the presence of the molecule. Importantly, the interaction of compound 4 can be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5′ UTR. Cell-based anti-HIV assays indicated that 4 inhibits HIV-induced cytopathicity in T lymphocytes with an EC50 of 28 μM, while cytotoxicity was not observed at concentrations approaching 1 mM. PMID:24820959

  6. A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

    PubMed Central

    Helka, Blake-Joseph; Brennan, John D.

    2013-01-01

    Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays. PMID:24022739

  7. An extremely simple method for fabricating 3D protein microarrays with an anti-fouling background and high protein capacity.

    PubMed

    Lin, Zhifeng; Ma, Yuhong; Zhao, Changwen; Chen, Ruichao; Zhu, Xing; Zhang, Lihua; Yan, Xu; Yang, Wantai

    2014-07-21

    Protein microarrays have become vital tools for various applications in biomedicine and bio-analysis during the past decade. The intense requirements for a lower detection limit and industrialization in this area have resulted in a persistent pursuit to fabricate protein microarrays with a low background and high signal intensity via simple methods. Here, we report on an extremely simple strategy to create three-dimensional (3D) protein microarrays with an anti-fouling background and a high protein capacity by photo-induced surface sequential controlled/living graft polymerization developed in our lab. According to this strategy, "dormant" groups of isopropyl thioxanthone semipinacol (ITXSP) were first introduced to a polymeric substrate through ultraviolet (UV)-induced surface abstraction of hydrogen, followed by a coupling reaction. Under visible light irradiation, the ITXSP groups were photolyzed to initiate surface living graft polymerization of poly(ethylene glycol) methyl methacrylate (PEGMMA), thus introducing PEG brushes to the substrate to generate a full anti-fouling background. Due to the living nature of this graft polymerization, there were still ITXSP groups on the chain ends of the PEG brushes. Therefore, by in situ secondary living graft cross-linking copolymerization of glycidyl methacrylate (GMA) and polyethylene glycol diacrylate (PEGDA), we could finally plant height-controllable cylinder microarrays of a 3D PEG network containing reactive epoxy groups onto the PEG brushes. Through a commonly used reaction of amine and epoxy groups, the proteins could readily be covalently immobilized onto the microarrays. This delicate design aims to overcome two universal limitations in protein microarrays: a full anti-fouling background can effectively eliminate noise caused by non-specific absorption and a 3D reactive network provides a larger protein-loading capacity to improve signal intensity. The results of non-specific protein absorption tests

  8. Kinetic identification of protein ligands in a 51,200 small-molecule library using microarrays and a label-free ellipsometric scanner

    NASA Astrophysics Data System (ADS)

    Landry, James P.; Proudian, Andrew P.; Malovichko, Galina; Zhu, Xiangdong

    2013-02-01

    Drug discovery begins by identifying protein-small molecule binding pairs. Afterwards, binding kinetics and biofunctional assays are performed, to reduce candidates for further development. High-throughput screening, typically employing fluorescence, is widely used to find protein ligands in small-molecule libraries, but is rarely used for binding kinetics measurement because: (1) attaching fluorophores to proteins can alter kinetics and (2) most label-free technologies for kinetics measurement are inherently low-throughput and consume expensive sensing surfaces. We addressed this need with polarization-modulated ellipsometric scanning microscopes, called oblique-incidence reflectivity difference (OI-RD). Label-free ligand screening and kinetics measurement are performed simultaneously on small-molecule microarrays printed on relatively inexpensive isocyanate-functionalized glass slides. As a microarray is reacted, an OI-RD microscope tracks the change in surface-bound macromolecule density in real-time at every spot. We report progress applying OI-RD to screen purified proteins and virus particles against a 51,200-compound library from the National Cancer Institute. Four microarrays, each containing 12,800 library compounds, are installed in four flow cells in an automated OI-RD microscope. The slides are reacted serially, each giving 12,800 binding curves with ~30 sec time resolution. The entire library is kinetically screened against a single probe in ~14 hours and multiple probes can be reacted sequentially under automation. Real-time binding detection identifies both high-affinity and low-affinity (transient binding) interactions; fluorescence endpoint images miss the latter. OI-RD and microarrays together is a powerful high-throughput tool for early stage drug discovery and development. The platform also has great potential for downstream steps such as in vitro inhibition assays.

  9. Calcium-binding proteins and development

    NASA Technical Reports Server (NTRS)

    Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

  10. Crossword: A Fully Automated Algorithm for the Segmentation and Quality Control of Protein Microarray Images

    PubMed Central

    2015-01-01

    Biological assays formatted as microarrays have become a critical tool for the generation of the comprehensive data sets required for systems-level understanding of biological processes. Manual annotation of data extracted from images of microarrays, however, remains a significant bottleneck, particularly for protein microarrays due to the sensitivity of this technology to weak artifact signal. In order to automate the extraction and curation of data from protein microarrays, we describe an algorithm called Crossword that logically combines information from multiple approaches to fully automate microarray segmentation. Automated artifact removal is also accomplished by segregating structured pixels from the background noise using iterative clustering and pixel connectivity. Correlation of the location of structured pixels across image channels is used to identify and remove artifact pixels from the image prior to data extraction. This component improves the accuracy of data sets while reducing the requirement for time-consuming visual inspection of the data. Crossword enables a fully automated protocol that is robust to significant spatial and intensity aberrations. Overall, the average amount of user intervention is reduced by an order of magnitude and the data quality is increased through artifact removal and reduced user variability. The increase in throughput should aid the further implementation of microarray technologies in clinical studies. PMID:24417579

  11. Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays.

    PubMed

    Coelho, Paulo S R; Im, Hogune; Clemons, Karl V; Snyder, Michael P; Stevens, David A

    2015-09-01

    We profiled the global immunoglobulin response against fungal infection by using yeast protein microarrays. Groups of CD-1 mice were infected systemically with human fungal pathogens (Coccidioides posadasii, Candida albicans, or Paracoccidioides brasiliensis) or inoculated with PBS as a control. Another group was inoculated with heat-killed yeast (HKY) of Saccharomyces cerevisiae. After 30 days, serum from mice in the groups were collected and used to probe S. cerevisiae protein microarrays containing 4800 full-length glutathione S-transferase (GST)-fusion proteins. Antimouse IgG conjugated with Alexafluor 555 and anti-GST antibody conjugated with Alexafluor 647 were used to detect antibody-antigen interactions and the presence of GST-fusion proteins, respectively. Serum after infection with C. albicans reacted with 121 proteins: C. posadasii, 81; P. brasiliensis, 67; and after HKY, 63 proteins on the yeast protein microarray, respectively. We identified a set of 16 antigenic proteins that were shared across the three fungal pathogens. These include retrotransposon capsid proteins, heat shock proteins, and mitochondrial proteins. Five of these proteins were identified in our previous study of fungal cell wall by mass spectrometry (Ann. N. Y. Acad. Sci. 2012, 1273, 44-51). The results obtained give a comprehensive view of the immunological responses to fungal infections at the proteomic level. They also offer insight into immunoreactive protein commonality among several fungal pathogens and provide a basis for a panfungal vaccine. PMID:26258609

  12. Backbone Dynamics Of Intracellular Lipid Binding Proteins

    NASA Astrophysics Data System (ADS)

    Gutiérrez-González, Luis H.

    2005-04-01

    The family of intracellular lipid binding proteins (iLBPs) comprises a group of homologous 14-15 kDa proteins that specifically bind and facilitate the transport of fatty acids, bile acids, retinoids or eicosanoids. Members of this family include several types of fatty acid binding proteins (FABPs), ileal lipid binding protein, cellular retinoic acid binding proteins and cellular retinoid binding proteins. As a contribution to understanding the structure-function relationship in this protein family, the solution structure and backbone dynamics of human epidermal-type FABP (E-FABP) determined by NMR spectroscopy are reported. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics discussed in the present study are compared with those obtained for other phylogenetically related proteins. A strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family is shown.

  13. A novel concept for protein microarray: land-contrast BioCD

    NASA Astrophysics Data System (ADS)

    Wang, Xuefeng; Zhao, Ming; Nolte, David D.

    2009-02-01

    We present a new type of protein microarray called the Land-contrast (LC) BioCD in which imaging contrast is induced by a patterned substrate rather than by patterned protein. This is realized by etching spot patterns in a silicon wafer. On the spot region the silicon dioxide thickness is 140 nm and on the land it is 77 nm. The spot and the land have equal reflectance but opposite interferometric quadrature responses for protein layer. Protein is evenly immobilized on the entire chip and detected by reflectometry. Therefore there is no need for protein printing, nor spectrometers, nor high angles nor polarization control to image the surface-bound protein. The LC BioCD can facilitate research on protein microarrays.

  14. The detection of DNA-binding proteins by protein blotting.

    PubMed Central

    Bowen, B; Steinberg, J; Laemmli, U K; Weintraub, H

    1980-01-01

    A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed. Images PMID:6243775

  15. Profiling Lipid–protein Interactions Using Nonquenched Fluorescent Liposomal Nanovesicles and Proteome Microarrays*

    PubMed Central

    Lu, Kuan-Yi; Tao, Sheng-Ce; Yang, Tzu-Ching; Ho, Yu-Hsuan; Lee, Chia-Hsien; Lin, Chen-Ching; Juan, Hsueh-Fen; Huang, Hsuan-Cheng; Yang, Chin-Yu; Chen, Ming-Shuo; Lin, Yu-Yi; Lu, Jin-Ying; Zhu, Heng; Chen, Chien-Sheng

    2012-01-01

    Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 μm of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid–protein

  16. Protein Microarrays: A New Tool for the Study of Autoantibodies in Immunodeficiency

    PubMed Central

    Rosenberg, Jacob M.; Utz, Paul J.

    2015-01-01

    Autoimmunity is highly coincident with immunodeficiency. In a small but growing number of primary immunodeficiencies, autoantibodies are diagnostic of a given disease and implicated in disease pathogenesis. In order to improve our understanding of the role of autoantibodies in immunodeficiencies and to discover novel autoantibodies, new proteomic tools are needed. Protein microarrays have the ability to screen for reactivity to hundreds to many thousands of unique autoantigens simultaneously on a single chip using minimal serum input. Here, we review different types of protein microarrays and how they can be useful in framing the study of primary and secondary immunodeficiencies. PMID:25904912

  17. Protein microarrays: a new tool for the study of autoantibodies in immunodeficiency.

    PubMed

    Rosenberg, Jacob M; Utz, Paul J

    2015-01-01

    Autoimmunity is highly coincident with immunodeficiency. In a small but growing number of primary immunodeficiencies, autoantibodies are diagnostic of a given disease and implicated in disease pathogenesis. In order to improve our understanding of the role of autoantibodies in immunodeficiencies and to discover novel autoantibodies, new proteomic tools are needed. Protein microarrays have the ability to screen for reactivity to hundreds to many thousands of unique autoantigens simultaneously on a single chip using minimal serum input. Here, we review different types of protein microarrays and how they can be useful in framing the study of primary and secondary immunodeficiencies. PMID:25904912

  18. Quantification of the Epitope Diversity of HIV-1-Specific Binding Antibodies by Peptide Microarrays for Global HIV-1 Vaccine Development

    PubMed Central

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-01-01

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6,564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research. PMID:25445329

  19. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    SciTech Connect

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.

  20. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    DOE PAGESBeta

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

  1. Haptenation: Chemical Reactivity and Protein Binding

    PubMed Central

    Chipinda, Itai; Hettick, Justin M.; Siegel, Paul D.

    2011-01-01

    Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed. PMID:21785613

  2. Calmodulin Binding Proteins and Alzheimer's Disease.

    PubMed

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  3. An overview of innovations and industrial solutions in Protein Microarray Technology.

    PubMed

    Gupta, Shabarni; Manubhai, K P; Kulkarni, Vishwesh; Srivastava, Sanjeeva

    2016-04-01

    The complexity involving protein array technology reflects in the fact that instrumentation and data analysis are subject to change depending on the biological question, technical compatibility of instruments and software used in each experiment. Industry has played a pivotal role in establishing standards for future deliberations in sustenance of these technologies in the form of protein array chips, arrayers, scanning devices, and data analysis software. This has enhanced the outreach of protein microarray technology to researchers across the globe. These have encouraged a surge in the adaptation of "nonclassical" approaches such as DNA-based protein arrays, micro-contact printing, label-free protein detection, and algorithms for data analysis. This review provides a unique overview of these industrial solutions available for protein microarray based studies. It aims at assessing the developments in various commercial platforms, thus providing a holistic overview of various modalities, options, and compatibility; summarizing the journey of this powerful high-throughput technology. PMID:27089056

  4. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    NASA Astrophysics Data System (ADS)

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  5. Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range

    PubMed Central

    Tabakman, Scott M.; Lau, Lana; Robinson, Joshua T.; Price, Jordan; Sherlock, Sarah P.; Wang, Hailiang; Zhang, Bo; Chen, Zhuo; Tangsombatvisit, Stephanie; Jarrell, Justin A.; Utz, Paul J.; Dai, Hongjie

    2012-01-01

    Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present protein microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of protein detection by three orders of magnitude towards the fM regime. We employ plasmonic protein microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic protein chips presents new opportunities in proteomic research and diagnostics applications. PMID:21915108

  6. Partial characterization of a proacrosin binding protein.

    PubMed

    Yi, L S; Runion, C M; Willand, J L; Polakoski, K L

    1992-01-01

    All of the acid (pH 4.0) extracted proacrosin from porcine epididymal spermatozoa was found to be tightly associated with a specific protein referred to as the binding protein. A combination of gel filterations and gel electrophoresis revealed that the binding protein is composed of a major 28 kd and a minor 29 kd protein. Both of the proteins were shown to be nonproteolytic by gelatin SDS-PAGE analysis and the amino acid composition analysis of the purified 28 kd protein revealed that it is not related to the proteolytic component of the proacrosinacrosin system. PMID:1519775

  7. Mercury-binding proteins of Mytilus edulis

    SciTech Connect

    Roesijadi, G.; Morris, J.E.; Calabrese, A.

    1981-11-01

    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 ..mu..g/l mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  8. Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing.

    PubMed

    2015-01-01

    The author's email has been corrected in the publication of Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to: j.zhou@uws.edu.au from: jzho7551@mail.usyd.edu.au. PMID:26167960

  9. Electrospinning of poly(dimethylsiloxane)/poly(methyl methacrylate) nanofibrous membrane: fabrication and application in protein microarrays.

    PubMed

    Yang, Dayong; Liu, Xing; Jin, Yu; Zhu, Ying; Zeng, Dongdong; Jiang, Xingyu; Ma, Hongwei

    2009-12-14

    Fabrication of poly(dimethylsiloxane) (PDMS)/poly(methyl methacrylate) (PMMA) nanofibers is critical to harness the advantage of nanostructured membrane applied in protein microarrays. Electrospinning (ES) of PDMS nanofibers is challenging because of the relatively low molecular weight of PDMS prepolymer. We report a strategy to fabricate PDMS/PMMA nanofibers via ES by introducing carrier polymer PMMA into PDMS solutions to supplement the deficiency of chain entanglements in the PDMS prepolymer. The prepared PDMS/PMMA nanofibrous membrane (PDMS/PMMA NFM) was successfully used as substrates for protein microarrays. The results of immunoassays showed the superior performance of PDMS/PMMA NFM as 3D substrate for protein microarrays; the limit-of-detection (LOD) on PDMS/PMMA NFM was 32 times lower than that on nitrocellulose membrane. The realization of ES PDMS extends the scope of ES materials from thermoplastic polymers to thermosetting materials. Given the simplicity, low cost, and high efficiency of ES technology, we believe that PDMS/PMMA NFM is a promising 3D substrate for protein microarrays. PMID:19924999

  10. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

    PubMed

    Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

    2015-09-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

  11. Aptamer Microarrays

    SciTech Connect

    Angel-Syrett, Heather; Collett, Jim; Ellington, Andrew D.

    2009-01-02

    In vitro selection can yield specific, high-affinity aptamers. We and others have devised methods for the automated selection of aptamers, and have begun to use these reagents for the construction of arrays. Arrayed aptamers have proven to be almost as sensitive as their solution phase counterparts, and when ganged together can provide both specific and general diagnostic signals for proteins and other analytes. We describe here technical details regarding the production and processing of aptamer microarrays, including blocking, washing, drying, and scanning. We will also discuss the challenges involved in developing standardized and reproducible methods for binding and quantitating protein targets. While signals from fluorescent analytes or sandwiches are typically captured, it has proven possible for immobilized aptamers to be uniquely coupled to amplification methods not available to protein reagents, thus allowing for protein-binding signals to be greatly amplified. Into the future, many of the biosensor methods described in this book can potentially be adapted to array formats, thus further expanding the utility of and applications for aptamer arrays.

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

    PubMed Central

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions. PMID:26540053

  13. The molecular architecture of protein-protein binding sites.

    PubMed

    Reichmann, Dana; Rahat, Ofer; Cohen, Mati; Neuvirth, Hani; Schreiber, Gideon

    2007-02-01

    The formation of specific protein interactions plays a crucial role in most, if not all, biological processes, including signal transduction, cell regulation, the immune response and others. Recent advances in our understanding of the molecular architecture of protein-protein binding sites, which facilitates such diversity in binding affinity and specificity, are enabling us to address key questions. What is the amino acid composition of binding sites? What are interface hotspots? How are binding sites organized? What are the differences between tight and weak interacting complexes? How does water contribute to binding? Can the knowledge gained be translated into protein design? And does a universal code for binding exist, or is it the architecture and chemistry of the interface that enable diverse but specific binding solutions? PMID:17239579

  14. Structure and Function of Lipopolysaccharide Binding Protein

    NASA Astrophysics Data System (ADS)

    Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

    1990-09-01

    The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

  15. Lamin-Binding Proteins in Caenorhabditis elegans.

    PubMed

    Dobrzynska, Agnieszka; Askjaer, Peter; Gruenbaum, Yosef

    2016-01-01

    The nuclear lamina, composed of lamins and numerous lamin-associated proteins, is required for mechanical stability, mechanosensing, chromatin organization, developmental gene regulation, mRNA transcription, DNA replication, nuclear assembly, and nuclear positioning. Mutations in lamins or lamin-binding proteins cause at least 18 distinct human diseases that affect specific tissues such as muscle, adipose, bone, nerve, or skin, and range from muscular dystrophies to lipodystrophy, peripheral neuropathy, or accelerated aging. Caenorhabditis elegans has unique advantages in studying lamin-binding proteins. These advantages include the low complexity of genes encoding lamin and lamin-binding proteins, advanced transgenic techniques, simple application of RNA interference, sophisticated genetic strategies, and a large collection of mutant lines. This chapter provides detailed and comprehensive protocols for the genetic and phenotypic analysis of lamin-binding proteins in C. elegans. PMID:26778571

  16. “On silico” peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions

    PubMed Central

    Price, Jordan V; Tangsombatvisit, Stephanie; Xu, Guangyu; Levy, Dan; Baechler, Emily C.; Gozani, Or; Varma, Madoo; Liu, Chih Long

    2011-01-01

    We have developed a novel, silicon-based peptide array for broad biological applications, including potential for development as a real-time point-of-care platform. We employed photolithography on silicon wafers to synthesize microarrays (Intel arrays), containing every possible overlapping peptide within a linear protein sequence covering the N-terminal tail of human histone H2B. Arrays also included peptides with acetylated and methylated lysine residues reflecting post-translational modifications of H2B. We defined minimum binding epitopes for commercial antibodies recognizing modified and unmodified H2B peptides. We further demonstrated that this platform is suitable for highly sensitive methyltransferase and kinase substrate characterization. Intel arrays also revealed specific H2B epitopes recognized by autoantibodies in individuals with systemic lupus erythematosus (SLE) that have increased disease severity. By combining emerging nonfluorescence-based detection methods with an underlying integrated circuit, we are now poised to create a truly transformative proteomics platform with applications in bioscience, drug development, and clinical diagnostics. PMID:22902875

  17. Microarray study of single nucleotide polymorphisms and expression of ATP-binding cassette genes in breast tumors

    NASA Astrophysics Data System (ADS)

    Tsyganov, M. M.; Ibragimova, M. K.; Karabut, I. V.; Freydin, M. B.; Choinzonov, E. L.; Litvyakov, N. V.

    2015-11-01

    Our previous research establishes that changes of expression of the ATP-binding cassette genes family is connected with the neoadjuvant chemotherapy effect. However, the mechanism of regulation of resistance gene expression remains unclear. As many researchers believe, single nucleotide polymorphisms can be involved in this process. Thereupon, microarray analysis is used to study polymorphisms in ATP-binding cassette genes. It is thus found that MDR gene expression is connected with 5 polymorphisms, i.e. rs241432, rs241429, rs241430, rs3784867, rs59409230, which participate in the regulation of expression of own genes.

  18. Robust microarray production of freshly expressed proteins in a human milieu

    PubMed Central

    Festa, Fernanda; Rollins, Sean M.; Vattem, Krishna; Hathaway, Margarita; Lorenz, Phillip; Mendoza, Eliseo Alejandro; Yu, Xiaobo; Qiu, Ji; Kilmer, Greg; Jensen, Penny; Webb, Brian; Ryan, Ed T.; LaBaer, Joshua

    2013-01-01

    Purpose In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu. Experimental design Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by 1-Step Human Coupled IVT (HCIVT) compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine. Results HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than 10 times higher than RRL, in both western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array. Conclusion and clinical relevance HCIVT is a robust IVTT system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer. PMID:23027544

  19. Towards zirconium phosphonate-based microarrays for probing DNA-protein interactions: critical influence of the location of the probe anchoring groups.

    PubMed

    Monot, Julien; Petit, Marc; Lane, Sarah M; Guisle, Isabelle; Léger, Jean; Tellier, Charles; Talham, Daniel R; Bujoli, Bruno

    2008-05-14

    Terminal phosphate groups on double-stranded DNA probes bind strongly to glass substrates coated with a zirconium phosphonate monolayer, and probes immobilized in this way as microarrays can be used to detect protein targets. The sensitivity of the microarray was shown to be enhanced by the use of a polyguanine segment ((G)n , n > or = 5) as a spacer between the phosphate linker and the protein interaction domain. More importantly, the presence of phosphate linkers on both ends of the dsDNA probes leads to significant enhancement of target capture. The relevant characteristics of the different probes when bound to the surface were determined, by the original use of a combination of surface characterization techniques (XPS, AFM, and Sarfus). In this context, the location of the phosphate linkers in the duplex probes was found to result in different probe surface coverage and presentation on the surface, which affect subsequent interactions with the target protein. PMID:18407629

  20. Evolution of Protein Binding Modes in Homooligomers

    PubMed Central

    Dayhoff, Judith E.; Shoemaker, Benjamin A.; Bryant, Stephen H.; Panchenko, Anna R.

    2009-01-01

    The evolution of protein interactions cannot be deciphered without a detailed analysis of interaction interfaces and binding modes. We performed a large-scale study of protein homooligomers in terms of their symmetry, interface sizes, and conservation of binding modes. We also focused specifically on the evolution of protein binding modes from nine families of homooligomers and mapped 60 different binding modes and oligomerization states onto the phylogenetic trees of these families. We observed a significant tendency for the same binding modes to be clustered together and conserved within clades on phylogenetic trees; this trend is especially pronounced for close homologs with 70% sequence identity or higher. Some binding modes are conserved among very distant homologs, pointing to their ancient evolutionary origin, while others are very specific for a certain phylogenetic group. Moreover, we found that the most ancient binding modes have a tendency to involve symmetrical (isologous) homodimer binding arrangements with larger interfaces, while recently evolved binding modes more often exhibit asymmetrical arrangements and smaller interfaces. PMID:19879880

  1. A Chemiluminescent Protein Microarray Method for Determining the Seroglycoid Fucosylation Index

    PubMed Central

    Zhang, Aiying; Skog, Sven; Wang, Shengqi; Ke, Yang; Zhang, Yonghong; Li, Kang; He, Ellen; Li, Ning

    2016-01-01

    The Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) is widely used to screen for hepatocellular carcinoma (HCC) in Japan and China. We developed a chemiluminescent protein microarray for determining the AFP-L3/AFP index (the ratio of AFP-L3 to total AFP, AFP-L3%) by fixing AFP-specific antibodies and Lens culinaris lectin on aldehyde-coated glass slides. Serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA) to validate the microarray. AFP-L3 was detected using Hotgen Biotech glycosyl capture spin column pretreatment technology and ELISA. When the AFP cut-off value was set to 20 ng/ml, the protein microarray displayed 89.74% sensitivity and 100% specificity for HCC diagnosis, and the ELISA displayed 87.17% sensitivity and 100% specificity. When the AFP-L3% cut-off value was set to 0.1, the protein microarray displayed 56.41% sensitivity and 100% specificity for HCC diagnosis, and the ELISA displayed 53.84% sensitivity and 100% specificity. The ROC curve for the HCC diagnosis showed that the AFP area under the ROC curve (AUC = 0.996; 95% CI: 0.986–1.005) was much higher than that of AFP-L3 (AUC = 0.857; 95% CI: 0.769–0.94) and AFP-L3% (AUC = 0.827; CI: 0.730–0.924). The microarray assay used in this study is a highly sensitive, accurate, and efficient assay for the determination of the AFP-L3%. PMID:27528397

  2. A Chemiluminescent Protein Microarray Method for Determining the Seroglycoid Fucosylation Index.

    PubMed

    Zhang, Aiying; Skog, Sven; Wang, Shengqi; Ke, Yang; Zhang, Yonghong; Li, Kang; He, Ellen; Li, Ning

    2016-01-01

    The Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) is widely used to screen for hepatocellular carcinoma (HCC) in Japan and China. We developed a chemiluminescent protein microarray for determining the AFP-L3/AFP index (the ratio of AFP-L3 to total AFP, AFP-L3%) by fixing AFP-specific antibodies and Lens culinaris lectin on aldehyde-coated glass slides. Serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA) to validate the microarray. AFP-L3 was detected using Hotgen Biotech glycosyl capture spin column pretreatment technology and ELISA. When the AFP cut-off value was set to 20 ng/ml, the protein microarray displayed 89.74% sensitivity and 100% specificity for HCC diagnosis, and the ELISA displayed 87.17% sensitivity and 100% specificity. When the AFP-L3% cut-off value was set to 0.1, the protein microarray displayed 56.41% sensitivity and 100% specificity for HCC diagnosis, and the ELISA displayed 53.84% sensitivity and 100% specificity. The ROC curve for the HCC diagnosis showed that the AFP area under the ROC curve (AUC = 0.996; 95% CI: 0.986-1.005) was much higher than that of AFP-L3 (AUC = 0.857; 95% CI: 0.769-0.94) and AFP-L3% (AUC = 0.827; CI: 0.730-0.924). The microarray assay used in this study is a highly sensitive, accurate, and efficient assay for the determination of the AFP-L3%. PMID:27528397

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

    PubMed

    Swaffield, J C; Johnston, S A

    2001-05-01

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

  4. Integrated Protein Array Screening and High Throughput Validation of 70 Novel Neural Calmodulin-binding Proteins*

    PubMed Central

    O'Connell, David J.; Bauer, Mikael C.; O'Brien, John; Johnson, Winifred M.; Divizio, Catherine A.; O'Kane, Sara L.; Berggård, Tord; Merino, Alejandro; Åkerfeldt, Karin S.; Linse, Sara; Cahill, Dolores J.

    2010-01-01

    Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca2+ ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca2+. This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (KD ≤ 1 μm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (koff ≤ 10−3 s−1) and high affinity (KD ≤ 1 μm), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We developed a microarray of the identified target proteins with which we can characterize the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage-gated channel Kv6.1 (residues 474–493), calmodulin kinase-like vesicle-associated protein (residues 302–316), EF-hand domain family member A2 (residues 202–216), and phosphatidylinositol-4-phosphate 5-kinase, type I, γ (residues 400–415). PMID:20068228

  5. Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone

    PubMed Central

    Ludwig, Susann K. J.; Tokarski, Christian; Lang, Stefan N.; van Ginkel, Leendert A.; Zhu, Hongying; Ozcan, Aydogan; Nielen, Michel W. F.

    2015-01-01

    Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this ‘protein microarray on a smartphone’-concept for on-site testing, e.g., in food safety, environment and health monitoring. PMID:26308444

  6. Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone.

    PubMed

    Ludwig, Susann K J; Tokarski, Christian; Lang, Stefan N; van Ginkel, Leendert A; Zhu, Hongying; Ozcan, Aydogan; Nielen, Michel W F

    2015-01-01

    Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this 'protein microarray on a smartphone'-concept for on-site testing, e.g., in food safety, environment and health monitoring. PMID:26308444

  7. Copper binding in the prion protein.

    PubMed

    Millhauser, Glenn L

    2004-02-01

    A conformational change of the prion protein is responsible for a class of neurodegenerative diseases called the transmissible spongiform encephalopathies that include mad cow disease and the human afflictions kuru and Creutzfeldt-Jakob disease. Despite the attention given to these diseases, the normal function of the prion protein in healthy tissue is unknown. Research over the past few years, however, demonstrates that the prion protein is a copper binding protein with high selectivity for Cu(2+). The structural features of the Cu(2+) binding sites have now been characterized and are providing important clues about the normal function of the prion protein and perhaps how metals or loss of protein function play a role in disease. The link between prion protein and copper may provide insight into the general, and recently appreciated, role of metals in neurodegenerative disease. PMID:14967054

  8. Microelectrospotting as a new method for electrosynthesis of surface-imprinted polymer microarrays for protein recognition.

    PubMed

    Bosserdt, Maria; Erdőssy, Júlia; Lautner, Gergely; Witt, Julia; Köhler, Katja; Gajovic-Eichelmann, Nenad; Yarman, Aysu; Wittstock, Gunther; Scheller, Frieder W; Gyurcsányi, Róbert E

    2015-11-15

    Here we introduce microelectrospotting as a new approach for preparation of protein-selective molecularly imprinted polymer microarrays on bare gold SPR imaging chips. During electrospotting both the gold chip and the spotting tip are electrically connected to a potentiostat as working and counter electrodes, respectively. The spotting pin encloses the monomer-template protein cocktail that upon contacting the gold surface is in-situ electropolymerized resulting in surface confined polymer spots of ca. 500 µm diameter. By repeating this procedure at preprogrammed locations for various composition monomer-template mixtures microarrays of nanometer-thin surface-imprinted films are generated in a controlled manner. We show that the removal and rebinding kinetics of the template and various potential interferents to such microarrays can be monitored in real-time and multiplexed manner by SPR imaging. The proof of principle for microelectrospotting of electrically insulating surface-imprinted films is made by using scopoletin as monomer and ferritin as protein template. It is shown that microelectrospotting in combination with SPR imaging can offer a versatile platform for label-free and enhanced throughput optimization of the molecularly imprinted polymers for protein recognition and for their analytical application. PMID:26056955

  9. Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

    PubMed Central

    Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

    2015-01-01

    A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

  10. Phosphoprotein Stability in Clinical Tissue and Its Relevance for Reverse Phase Protein Microarray Technology

    PubMed Central

    Espina, Virginia; Mueller, Claudius; Liotta, Lance A.

    2013-01-01

    Phosphorylated proteins reflect the activity of specific cell signaling nodes in biological kinase protein networks. Cell signaling pathways can be either activated or deactivated depending on the phosphorylation state of the constituent proteins. The state of these kinase pathways reflects the in vivo activity of the cells and tissue at any given point in time. As such, cell signaling pathway information can be extrapolated to infer which phosphorylated proteins/pathways are driving an individual tumor’s growth. Reverse Phase Protein Microarrays (RPMA) are a sensitive and precise platform that can be applied to the quantitative measurement of hundreds of phosphorylated signal proteins from a small sample of tissue. Pre-analytical variability originating from tissue procurement and preservation may cause significant variability and bias in downstream molecular analysis. Depending on the ex vivo delay time in tissue processing, and the manner of tissue handling, protein biomarkers such as signal pathway phosphoproteins will be elevated or suppressed in a manner that does not represent the biomarker levels at the time of excision. Consequently, assessment of the state of these kinase networks requires stabilization, or preservation, of the phosphoproteins immediately post tissue procurement. We have employed reverse phase protein microarray analysis of phosphoproteins to study the factors influencing stability of phosphoproteins in tissue following procurement. Based on this analysis we have established tissue procurement guidelines for clinical research with an emphasis on quantifying phosphoproteins by RPMA. PMID:21901591

  11. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

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

    PubMed Central

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

    2016-01-01

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

  13. Deregulated Direct Targets of the Hepatitis B Virus (HBV) Protein, HBx, Identified through Chromatin Immunoprecipitation and Expression Microarray Profiling*

    PubMed Central

    Sung, Wing-Kin; Lu, Yiwei; Lee, Charlie W. H.; Zhang, Dongwei; Ronaghi, Mostafa; Lee, Caroline G. L.

    2009-01-01

    The hepatitis B-X (HBx) protein is strongly associated with hepatocellular carcinoma. It is implicated not to directly cause cancer but to play a role in hepatocellular carcinoma as a co-factor. The oncogenic potential of HBx primarily lies in its interaction with transcriptional regulators resulting in aberrant gene expression and deregulated cellular pathways. Utilizing ultraviolet irradiation to simulate a tumor-initiating event, we integrated chip-based chromatin immunoprecipitation (ChIP-chip) with expression microarray profiling and identified 184 gene targets directly deregulated by HBx. One-hundred forty-four transcription factors interacting with HBx were computationally inferred. We experimentally validated that HBx interacts with some of the predicted transcription factors (pTF) as well as the promoters of the deregulated target genes of these pTFs. Significantly, we demonstrated that the pTF interacts with the promoters of the deregulated HBx target genes and that deregulation by HBx of these HBx target genes carrying the pTF consensus sequences can be reversed using pTF small interfering RNAs. The roles of these deregulated direct HBx target genes and their relevance in cancer was inferred via querying against biogroup/cancer-related microarray databases using web-based NextBioTM software. Six pathways, including the Jak-STAT pathway, were predicted to be significantly deregulated when HBx binds indirectly to direct target gene promoters. In conclusion, this study represents the first ever demonstration of the utilization of ChIP-chip to identify deregulated direct gene targets from indirect protein-DNA binding as well as transcriptional factors directly interacting with HBx. Increased knowledge of the gene/transcriptional factor targets of HBx will enhance our understanding of the role of HBx in hepatocellular carcinogenesis and facilitate the design of better strategies in combating hepatitis B virus-associated hepatocellular carcinoma. PMID:19439406

  14. Deregulated direct targets of the hepatitis B virus (HBV) protein, HBx, identified through chromatin immunoprecipitation and expression microarray profiling.

    PubMed

    Sung, Wing-Kin; Lu, Yiwei; Lee, Charlie W H; Zhang, Dongwei; Ronaghi, Mostafa; Lee, Caroline G L

    2009-08-14

    The hepatitis B-X (HBx) protein is strongly associated with hepatocellular carcinoma. It is implicated not to directly cause cancer but to play a role in hepatocellular carcinoma as a co-factor. The oncogenic potential of HBx primarily lies in its interaction with transcriptional regulators resulting in aberrant gene expression and deregulated cellular pathways. Utilizing ultraviolet irradiation to simulate a tumor-initiating event, we integrated chip-based chromatin immunoprecipitation (ChIP-chip) with expression microarray profiling and identified 184 gene targets directly deregulated by HBx. One-hundred forty-four transcription factors interacting with HBx were computationally inferred. We experimentally validated that HBx interacts with some of the predicted transcription factors (pTF) as well as the promoters of the deregulated target genes of these pTFs. Significantly, we demonstrated that the pTF interacts with the promoters of the deregulated HBx target genes and that deregulation by HBx of these HBx target genes carrying the pTF consensus sequences can be reversed using pTF small interfering RNAs. The roles of these deregulated direct HBx target genes and their relevance in cancer was inferred via querying against biogroup/cancer-related microarray databases using web-based NextBio(TM) software. Six pathways, including the Jak-STAT pathway, were predicted to be significantly deregulated when HBx binds indirectly to direct target gene promoters. In conclusion, this study represents the first ever demonstration of the utilization of ChIP-chip to identify deregulated direct gene targets from indirect protein-DNA binding as well as transcriptional factors directly interacting with HBx. Increased knowledge of the gene/transcriptional factor targets of HBx will enhance our understanding of the role of HBx in hepatocellular carcinogenesis and facilitate the design of better strategies in combating hepatitis B virus-associated hepatocellular carcinoma. PMID

  15. Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

    PubMed

    Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

    2013-01-01

    Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB. PMID:24145242

  16. A computational framework for the analysis of peptide microarray antibody binding data with application to HIV vaccine profiling.

    PubMed

    Imholte, Greg C; Sauteraud, Renan; Korber, Bette; Bailer, Robert T; Turk, Ellen T; Shen, Xiaoying; Tomaras, Georgia D; Mascola, John R; Koup, Richard A; Montefiori, David C; Gottardo, Raphael

    2013-09-30

    We present an integrated analytical method for analyzing peptide microarray antibody binding data, from normalization through subject-specific positivity calls and data integration and visualization. Current techniques for the normalization of such data sets do not account for non-specific binding activity. A novel normalization technique based on peptide sequence information quickly and effectively reduced systematic biases. We also employed a sliding mean window technique that borrows strength from peptides sharing similar sequences, resulting in reduced signal variability. A smoothed signal aided in the detection of weak antibody binding hotspots. A new principled FDR method of setting positivity thresholds struck a balance between sensitivity and specificity. In addition, we demonstrate the utility and importance of using baseline control measurements when making subject-specific positivity calls. Data sets from two human clinical trials of candidate HIV-1 vaccines were used to validate the effectiveness of our overall computational framework. PMID:23770318

  17. High performance protein microarrays based on glycidyl methacrylate-modified polyethylene terephthalate plastic substrate.

    PubMed

    Liu, Yingshuai; Li, Chang Ming; Hu, Weihua; Lu, Zhisong

    2009-01-15

    There is a great challenge to immobilize high density of probe molecules for high performance protein microarrays, and this is achieved in this work by using polyethylene terephthalate (PET) plastic substrate onto which glycidyl methacrylate (GMA) photopolymer is grafted under mild conditions to introduce high density of epoxy groups for covalent immobilization of proteins. The poly(GMA)-grafted PET (PGMA-PET) surface was characterized with atomic force microscope (AFM) and attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy. For high density of protein immobilization and good quality of microspots, experiments were conducted to optimize the printing buffer, and an optimal buffer was found out to be PBS with 10% glycerol+0.003% triton X-100. According to the studies of loading capacity and immobilization kinetics, the optimal protein probe concentration and incubation time for the efficient immobilization are 200 microg mL(-1) and 8h, respectively. The performance of the PGMA-PET-based protein microarrays is evaluated with sandwich immunoassay using rat IgG and anti-rat IgG as model proteins, demonstrating a limit of detection (LOD) of 10 pg mL(-1) and a dynamic range of five orders of magnitude which are better than or very comparable with the reported or commercially available immunoassays, while providing a high-throughput approach. The work renders a simple and economic method to manufacture high performance protein microarrays and is expected to have great potentials in broad applications related to clinic diagnosis, drug discovery and proteomic research. PMID:19064107

  18. Astaxanthin binding protein in Atlantic salmon.

    PubMed

    Matthews, Sarah J; Ross, Neil W; Lall, Santosh P; Gill, Tom A

    2006-06-01

    The rubicund pigmentation in salmon and trout flesh is unique and is due to the deposition of dietary carotenoids, astaxanthin and canthaxanthin in the muscle. The present study was undertaken to determine which protein was responsible for pigment binding. Salmon muscle proteins were solubilized by sequential extractions with non-denaturing, low ionic strength aqueous solutions and segregated as such into six different fractions. Approximately 91% of the salmon myofibrillar proteins were solubilized under non-denaturing conditions using a protocol modified from a method described by Krishnamurthy et al. [Krishnamurthy, G., Chang, H.S., Hultin, H.O., Feng, Y., Srinivasan, S., Kelleher. S.D., 1996. Solubility of chicken breast muscle proteins in solutions of low ionic strength. J. Agric. Food Chem. 44: 408-415.] for the dissolution of avian muscle. To our knowledge, this is the first time this solubilization approach has been applied to the study of molecular interactions in myofibrillar proteins. Astaxanthin binding in each fraction was determined using an in vitro binding assay. In addition, SDS-PAGE and quantitative densitometry were used to separate and determine the relative amounts of each of the proteins in the six fractions. The results showed that alpha-actinin was the only myofibrillar protein correlating significantly (P<0.05) with astaxanthin binding. Alpha-actinin was positively identified using electrophoretic techniques and confirmed by tandem mass spectroscopy. Purified salmon alpha-actinin bound synthetic astaxanthin in a molar ratio of 1.11:1.00. The study was repeated using halibut alpha-actinin, which was found to have a molar binding ratio of astaxanthin to alpha-actinin of 0.893:1. These results suggest that the difference in pigmentation between white fish and Atlantic salmon is not due to binding capacity in the muscle, but rather differences in the metabolism or transport of pigment. PMID:16644255

  19. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins.

    PubMed

    Clifton, Ben E; Jackson, Colin J

    2016-02-18

    The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity. PMID:26853627

  20. Aspects of Protein, Chemistry, Part II: Oxygen-Binding Proteins

    ERIC Educational Resources Information Center

    Nixon, J. E.

    1977-01-01

    Compares differences in function and behavior of two oxygen-binding proteins, myoglobin found in muscle and hemoglobin found in blood. Describes the mechanism of oxygen-binding and allosteric effect in hemoglobin; also describes the effect of pH on the affinity of hemoglobin for oxygen. (CS)

  1. Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos)

    PubMed Central

    Carlson, Ruth I.; Cattet, Marc R. L.; Sarauer, Bryan L.; Nielsen, Scott E.; Boulanger, John; Stenhouse, Gordon B.; Janz, David M.

    2016-01-01

    A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic–pituitary–adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50–100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally <10 and <15%, respectively. With one exception, there were no significant differences in protein expression among skin samples collected from the neck, forelimb, hindlimb and ear in a subsample of n = 4 bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change. PMID:27293753

  2. Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).

    PubMed

    Carlson, Ruth I; Cattet, Marc R L; Sarauer, Bryan L; Nielsen, Scott E; Boulanger, John; Stenhouse, Gordon B; Janz, David M

    2016-01-01

    A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally <10 and <15%, respectively. With one exception, there were no significant differences in protein expression among skin samples collected from the neck, forelimb, hindlimb and ear in a subsample of n = 4 bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change. PMID:27293753

  3. Predicting Ca(2+)-binding sites in proteins.

    PubMed Central

    Nayal, M; Di Cera, E

    1994-01-01

    The coordination shell of Ca2+ ions in proteins contains almost exclusively oxygen atoms supported by an outer shell of carbon atoms. The bond-strength contribution of each ligating oxygen in the inner shell can be evaluated by using an empirical expression successfully applied in the analysis of crystals of metal oxides. The sum of such contributions closely approximates the valence of the bound cation. When a protein is embedded in a very fine grid of points and an algorithm is used to calculate the valence of each point representing a potential Ca(2+)-binding site, a typical distribution of valence values peaked around 0.4 is obtained. In 32 documented Ca(2+)-binding proteins, containing a total of 62 Ca(2+)-binding sites, a very small fraction of points in the distribution has a valence close to that of Ca2+. Only 0.06% of the points have a valence > or = 1.4. These points share the remarkable tendency to cluster around documented Ca2+ ions. A high enough value of the valence is both necessary (58 out of 62 Ca(2+)-binding sites have a valence > or = 1.4) and sufficient (87% of the grid points with a valence > or = 1.4 are within 1.0 A from a documented Ca2+ ion) to predict the location of bound Ca2+ ions. The algorithm can also be used for the analysis of other cations and predicts the location of Mg(2+)- and Na(+)-binding sites in a number of proteins. The valence is, therefore, a tool of pinpoint accuracy for locating cation-binding sites, which can also be exploited in engineering high-affinity binding sites and characterizing the linkage between structural components and functional energetics for molecular recognition of metal ions by proteins. Images Fig. 4 PMID:8290605

  4. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    in vitro TF-DNA preferences obtained from the universal protein binding microarrays (PBM) for ~90 eukaryotic TFs belonging to 22 different DNA-binding domain types. As a result of this new analysis, we conclude that nonconsensus protein-DNA binding is a widespread phenomenon that significantly affects protein-DNA binding preferences and need not require the presence of consensus (specific) TFBSs in order to achieve genome-wide TF-DNA binding specificity. PMID:26285121

  5. Ice-Binding Proteins and Their Function.

    PubMed

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-01

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities. PMID:27145844

  6. Predicting protein concentrations with ELISA microarray assays, monotonic splines and Monte Carlo simulation

    SciTech Connect

    Daly, Don S.; Anderson, Kevin K.; White, Amanda M.; Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2008-07-14

    Background: A microarray of enzyme-linked immunosorbent assays, or ELISA microarray, predicts simultaneously the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Making sound biological inferences as well as improving the ELISA microarray process require require both concentration predictions and creditable estimates of their errors. Methods: We present a statistical method based on monotonic spline statistical models, penalized constrained least squares fitting (PCLS) and Monte Carlo simulation (MC) to predict concentrations and estimate prediction errors in ELISA microarray. PCLS restrains the flexible spline to a fit of assay intensity that is a monotone function of protein concentration. With MC, both modeling and measurement errors are combined to estimate prediction error. The spline/PCLS/MC method is compared to a common method using simulated and real ELISA microarray data sets. Results: In contrast to the rigid logistic model, the flexible spline model gave credible fits in almost all test cases including troublesome cases with left and/or right censoring, or other asymmetries. For the real data sets, 61% of the spline predictions were more accurate than their comparable logistic predictions; especially the spline predictions at the extremes of the prediction curve. The relative errors of 50% of comparable spline and logistic predictions differed by less than 20%. Monte Carlo simulation rendered acceptable asymmetric prediction intervals for both spline and logistic models while propagation of error produced symmetric intervals that diverged unrealistically as the standard curves approached horizontal asymptotes. Conclusions: The spline/PCLS/MC method is a flexible, robust alternative to a logistic/NLS/propagation-of-error method to reliably predict protein concentrations and estimate their errors. The spline method simplifies model selection and fitting

  7. Identification of the Feline Humoral Immune Response to Bartonella henselae Infection by Protein Microarray

    PubMed Central

    Vigil, Adam; Ortega, Rocio; Jain, Aarti; Nakajima-Sasaki, Rie; Tan, Xiaolin; Chomel, Bruno B.; Kasten, Rickie W.; Koehler, Jane E.; Felgner, Philip L.

    2010-01-01

    Background Bartonella henselae is the zoonotic agent of cat scratch disease and causes potentially fatal infections in immunocompromised patients. Understanding the complex interactions between the host's immune system and bacterial pathogens is central to the field of infectious diseases and to the development of effective diagnostics and vaccines. Methodology We report the development of a microarray comprised of proteins expressed from 96% (1433/1493) of the predicted ORFs encoded by the genome of the zoonotic pathogen Bartonella henselae. The array was probed with a collection of 62 uninfected, 62 infected, and 8 “specific-pathogen free” naïve cat sera, to profile the antibody repertoire elicited during natural Bartonella henselae infection. Conclusions We found that 7.3% of the B. henselae proteins on the microarray were seroreactive and that seroreactivity was not evenly distributed between predicted protein function or subcellular localization. Membrane proteins were significantly most likely to be seroreactive, although only 23% of the membrane proteins were reactive. Conversely, we found that proteins involved in amino acid transport and metabolism were significantly underrepresented and did not contain any seroreactive antigens. Of all seroreactive antigens, 52 were differentially reactive with sera from infected cats, and 53 were equally reactive with sera from infected and uninfected cats. Thirteen of the seroreactive antigens were found to be differentially seroreactive between B. henselae type I and type II. Based on these results, we developed a classifier algorithm that was capable of accurately discerning 93% of the infected animals using the microarray platform. The seroreactivity and diagnostic potential of these antigens was then validated on an immunostrip platform, which correctly identified 98% of the infected cats. Our protein microarray platform provides a high-throughput, comprehensive analysis of the feline humoral immune response to

  8. Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function.

    PubMed

    Kierzek, Elzbieta; Kierzek, Ryszard; Moss, Walter N; Christensen, Shawn M; Eickbush, Thomas H; Turner, Douglas H

    2008-04-01

    LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2' oxygen and 4' carbon of ribose) and 2,6-diaminopurine were incorporated into 2'-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5' coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5' RNA). This R2Bm 5' RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5' RNA. PMID:18252773

  9. Chemistry of Natural Glycan Microarray

    PubMed Central

    Song, Xuezheng; Heimburg-Molinaro, Jamie; Cummings, Richard D.; Smith, David F.

    2014-01-01

    Glycan microarrays have become indispensable tools for studying protein-glycan interactions. Along with chemo-enzymatic synthesis, glycans isolated from natural sources have played important roles in array development and will continue to be a major source of glycans. N- and O-glycans from glycoproteins, and glycans from glycosphingolipids can be released from corresponding glycoconjugates with relatively mature methods, although isolation of large numbers and quantities of glycans are still very challenging. Glycosylphosphatidylinositol (GPI)-anchors and glycosaminoglycans (GAGs) are less represented on current glycan microarrays. Glycan microarray development has been greatly facilitated by bifunctional fluorescent linkers, which can be applied in a “Shotgun Glycomics” approach to incorporate isolated natural glycans. Glycan presentation on microarrays may affect glycan binding by GBPs, often through multivalent recognition by the GBP. PMID:24487062

  10. Stretching DNA to quantify nonspecific protein binding

    NASA Astrophysics Data System (ADS)

    Goyal, Sachin; Fountain, Chandler; Dunlap, David; Family, Fereydoon; Finzi, Laura

    2012-07-01

    Nonspecific binding of regulatory proteins to DNA can be an important mechanism for target search and storage. This seems to be the case for the lambda repressor protein (CI), which maintains lysogeny after infection of E. coli. CI binds specifically at two distant regions along the viral genome and induces the formation of a repressive DNA loop. However, single-molecule imaging as well as thermodynamic and kinetic measurements of CI-mediated looping show that CI also binds to DNA nonspecifically and that this mode of binding may play an important role in maintaining lysogeny. This paper presents a robust phenomenological approach using a recently developed method based on the partition function, which allows calculation of the number of proteins bound nonspecific to DNA from measurements of the DNA extension as a function of applied force. This approach was used to analyze several cycles of extension and relaxation of λ DNA performed at several CI concentrations to measure the dissociation constant for nonspecific binding of CI (˜100 nM), and to obtain a measurement of the induced DNA compaction (˜10%) by CI.

  11. Signal transduction by guanine nucleotide binding proteins.

    PubMed

    Spiegel, A M

    1987-01-01

    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  12. [Carbohydrate-binding proteins of marine invertebrates].

    PubMed

    Luk'ianov, P A; Chernikov, O V; Kobelev, S S; Chikalovets, I V; Molchanova, V I; Li, W

    2007-01-01

    The information on the carbohydrate specificity and molecular organization of some carbohydrate-binding proteins (lectins) of marine invertebrates is reported. Antiviral activity of some of the lectins against human immunodeficiency virus has been studied. Lectins of marine invertebrates are promising tools for studying natural glycoconjugates and cell effectors in vitro. PMID:17375673

  13. Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay

    PubMed Central

    Joo, Chulmin; Özkumur, Emre; Ünlü, M. Selim; de Boer, Johannes F.

    2009-01-01

    Quantitative measurement of affinities and kinetics of various biomolecular interactions such as protein-protein, protein-DNA and receptor-ligand is central to our understanding of basic molecular and cellular functions and is useful for therapeutic evaluation. Here, we describe a laser-scanning quantitative imaging method, referred to as spectral-domain optical coherence phase microscopy, as an optical platform for label-free detection of biomolecular interactions. The instrument is based on a confocal interferometric microscope that enables depth-resolved quantitative phase measurements on sensor surface with high spatial resolution and phase stability. We demonstrate picogram per square millimeter surface mass sensitivity, and show its sensing capability by presenting static and dynamic detection of multiplexed protein microarray as immobilized antigens capture their corresponding antibodies. PMID:19674885

  14. Evolution of Protein-binding DNA Sequences through Competitive Binding

    NASA Astrophysics Data System (ADS)

    Peng, Weiqun; Gerland, Ulrich; Hwa, Terence; Levine, Herbert

    2002-03-01

    The dynamics of in vitro DNA evolution controlled via competitive binding of DNA sequences to proteins has been explored in a recent serial transfer experiment footnote B. Dubertret, S.Liu, Q. Ouyang, A. Libchaber, Phys. Rev. Lett. 86, 6022 (2001).. Motivated by the experiment, we investigate a continuum model for this evolution process in various parameter regimes. We establish a self-consistent mean-field evolution equation, determine its dynamical properties and finite population size corrections. In addition, we discuss the experimental implications of our results.

  15. Odorant-binding proteins in insects.

    PubMed

    Zhou, Jing-Jiang

    2010-01-01

    Our understanding of the molecular and biochemical mechanisms that mediate chemoreception in insects has been greatly improved after the discovery of olfactory and taste receptor proteins. However, after 50 years of the discovery of first insect sex pheromone from the silkmoth Bombyx mori, it is still unclear how hydrophobic compounds reach the dendrites of sensory neurons in vivo across aqueous space and interact with the sensory receptors. The presence of soluble polypeptides in high concentration in the lymph of chemosensilla still poses unanswered questions. More than two decades after their discovery and despite the wealth of structural and biochemical information available, the physiological function of odorant-binding proteins (OBPs) is not well understood. Here, I review the structural properties of different subclasses of insect OBPs and their binding to pheromones and other small ligands. Finally, I discuss current ideas and models on the role of such proteins in insect chemoreception. PMID:20831949

  16. Quantifying drug-protein binding in vivo.

    SciTech Connect

    Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

    2004-02-17

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS.

  17. Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements.

    PubMed

    Zhou, Heping; Bouwman, Kerri; Schotanus, Mark; Verweij, Cornelius; Marrero, Jorge A; Dillon, Deborah; Costa, Jose; Lizardi, Paul; Haab, Brian B

    2004-01-01

    The ability to conveniently and rapidly profile a diverse set of proteins has valuable applications. In a step toward further enabling such a capability, we developed the use of rolling-circle amplification (RCA) to measure the relative levels of proteins from two serum samples, labeled with biotin and digoxigenin, respectively, that have been captured on antibody microarrays. Two-color RCA produced fluorescence up to 30-fold higher than direct-labeling and indirect-detection methods using antibody microarrays prepared on both polyacrylamide-based hydrogels and nitrocellulose. Replicate RCA measurements of multiple proteins from sets of 24 serum samples were highly reproducible and accurate. In addition, RCA enabled reproducible measurements of distinct expression profiles from lower-abundance proteins that were not measurable using the other detection methods. Two-color RCA on antibody microarrays should allow the convenient acquisition of expression profiles from a great diversity of proteins for a variety of applications. PMID:15059261

  18. Microarrays, Integrated Analytical Systems

    NASA Astrophysics Data System (ADS)

    Combinatorial chemistry is used to find materials that form sensor microarrays. This book discusses the fundamentals, and then proceeds to the many applications of microarrays, from measuring gene expression (DNA microarrays) to protein-protein interactions, peptide chemistry, carbodhydrate chemistry, electrochemical detection, and microfluidics.

  19. A coprecipitation-based validation methodology for interactions identified using protein microarrays.

    PubMed

    Marina, Ovidiu; Duke-Cohan, Jonathan S; Wu, Catherine J

    2011-01-01

    Candidate interactions identified by high-throughput protein microarray screening require rigorous -confirmation. Such validation is time-consuming and labor-intensive using conventional techniques. We describe a medium-throughput validation protocol based on coprecipitation of biotin-labeled -proteins synthesized in vitro using a rabbit reticulocyte lysate-coupled transcription and translation system. As our experimental system is based on screening for serum antibodies, we also present methods on purifying immunoglobulin from serum and quantifying the amount of coprecipitated (immunoprecipitated) target protein on Western blot. This technique provides a sensitive confirmatory test allowing for the rapid elimination of false positives prior to more extensive validation and analysis of target interactions in their native environment. PMID:21370070

  20. Lectin microarray reveals binding profiles of Lactobacillus casei strains in a comprehensive analysis of bacterial cell wall polysaccharides.

    PubMed

    Yasuda, Emi; Tateno, Hiroaki; Hirabayashi, Jun; Hirabarashi, Jun; Iino, Tohru; Sako, Tomoyuki

    2011-07-01

    We previously showed a pivotal role of the polysaccharide (PS) moiety in the cell wall of the Lactobacillus casei strain Shirota (YIT 9029) as a possible immune modulator (E. Yasuda M. Serata, and T. Sako, Appl. Environ. Microbiol. 74:4746-4755, 2008). To distinguish PS structures on the bacterial cell surface of individual strains in relation to their activities, it would be useful to have a rapid and high-throughput methodology. Recently, a new technique called lectin microarray was developed for rapid profiling of glycosylation in eukaryotic polymers and cell surfaces. Here, we report on the development of a simple and sensitive method based on this technology for direct analysis of intact bacterial cell surface glycomes. The method involves labeling bacterial cells with SYTOX Orange before incubation with the lectin microarray. After washing, bound cells are directly detected using an evanescent-field fluorescence scanner in a liquid phase. Using this method, we compared the cell surface glycomes from 16 different strains of L. casei. The patterns of lectin-binding affinity of most strains were found to be unique. There appears to be two types of lectin-binding profiles: the first is characterized by a few lectins, and the other is characterized by multiple lectins with different specificities. We also showed a dramatic change in the lectin-binding profile of a YIT 9029 derivative with a mutation in the cps1C gene, encoding a putative glycosyltransferase. In conclusion, the developed technique provided a novel strategy for rapid profiling and, more importantly, differentiating numerous bacterial strains with relevance to the biological functions of PS. PMID:21602390

  1. Lectin Microarray Reveals Binding Profiles of Lactobacillus casei Strains in a Comprehensive Analysis of Bacterial Cell Wall Polysaccharides▿†

    PubMed Central

    Yasuda, Emi; Tateno, Hiroaki; Hirabarashi, Jun; Iino, Tohru; Sako, Tomoyuki

    2011-01-01

    We previously showed a pivotal role of the polysaccharide (PS) moiety in the cell wall of the Lactobacillus casei strain Shirota (YIT 9029) as a possible immune modulator (E. Yasuda M. Serata, and T. Sako, Appl. Environ. Microbiol. 74:4746-4755, 2008). To distinguish PS structures on the bacterial cell surface of individual strains in relation to their activities, it would be useful to have a rapid and high-throughput methodology. Recently, a new technique called lectin microarray was developed for rapid profiling of glycosylation in eukaryotic polymers and cell surfaces. Here, we report on the development of a simple and sensitive method based on this technology for direct analysis of intact bacterial cell surface glycomes. The method involves labeling bacterial cells with SYTOX Orange before incubation with the lectin microarray. After washing, bound cells are directly detected using an evanescent-field fluorescence scanner in a liquid phase. Using this method, we compared the cell surface glycomes from 16 different strains of L. casei. The patterns of lectin-binding affinity of most strains were found to be unique. There appears to be two types of lectin-binding profiles: the first is characterized by a few lectins, and the other is characterized by multiple lectins with different specificities. We also showed a dramatic change in the lectin-binding profile of a YIT 9029 derivative with a mutation in the cps1C gene, encoding a putative glycosyltransferase. In conclusion, the developed technique provided a novel strategy for rapid profiling and, more importantly, differentiating numerous bacterial strains with relevance to the biological functions of PS. PMID:21602390

  2. Cadmium-binding protein (metallothionein) in carp.

    PubMed Central

    Kito, H; Ose, Y; Sato, T

    1986-01-01

    When carp (Cyprinus carpio) were exposed to 5 and 30 ppm Cd in the water, the contents of Cd-binding protein, which has low molecular weight, increased in the hepatopancreas, kidney, gills and gastrointestinal tract with the duration of exposure. This Cd-binding protein was purified from hepatopancreas, kidney, gills, and spleen of carp administered 2 mg/kg Cd (as CdCl2), intraperitoneally for 6 days. Two Cd-binding proteins were separated by DEAE-Sephadex A-25 column chromatography. These proteins had Cd-mercaptide bond, high cysteine contents (ca. 29-34%), but no aromatic amino acids or histidine. From these characteristics the Cd-binding proteins were identified as metallothionein. By using antiserum obtained from a rabbit to which carp hepatopancreas MT-II had been administered, immunological characteristics between hepatopancreas MT-I, II and kidney MT-II were studied, and a slight difference in antigenic determinant was observed among them. By immunological staining techniques with horseradish peroxidase, the localization of metallothionein was investigated. In the nontreated group, metallothionein was present in the acinar cells of hepatopancreas and renal convoluted tubules. In the Cd-treated group (2 mg/kg IP daily for 3 days), metallothionein was present in the nuclei, sinusoids, and extracellular space of hepatopancreas, in addition to the acinar cells. Carp were bred in 1 ppm Cd, 5 ppm Zn solution, and tap water for 14 days, following transfer to 15 ppm Cd solution, respectively. The survival ratio was the highest in the Zn group followed by Cd-treated and control groups. The metallothionein contents increased in hepatopancreas and kidney in the order: Zn greater than Cd greater than control group. Images FIGURE 5. FIGURE 6. PMID:3519201

  3. Exploring host-pathogen interactions through genome wide protein microarray analysis.

    PubMed

    Scietti, Luigi; Sampieri, Katia; Pinzuti, Irene; Bartolini, Erika; Benucci, Barbara; Liguori, Alessia; Haag, Andreas F; Lo Surdo, Paola; Pansegrau, Werner; Nardi-Dei, Vincenzo; Santini, Laura; Arora, Seguinde; Leber, Xavier; Rindi, Simonetta; Savino, Silvana; Costantino, Paolo; Maione, Domenico; Merola, Marcello; Speziale, Pietro; Bottomley, Matthew J; Bagnoli, Fabio; Masignani, Vega; Pizza, Mariagrazia; Scharenberg, Meike; Schlaeppi, Jean-Marc; Nissum, Mikkel; Liberatori, Sabrina

    2016-01-01

    During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis. PMID:27302108

  4. Exploring host-pathogen interactions through genome wide protein microarray analysis

    PubMed Central

    Scietti, Luigi; Sampieri, Katia; Pinzuti, Irene; Bartolini, Erika; Benucci, Barbara; Liguori, Alessia; Haag, Andreas F.; Lo Surdo, Paola; Pansegrau, Werner; Nardi-Dei, Vincenzo; Santini, Laura; Arora, Seguinde; Leber, Xavier; Rindi, Simonetta; Savino, Silvana; Costantino, Paolo; Maione, Domenico; Merola, Marcello; Speziale, Pietro; Bottomley, Matthew J.; Bagnoli, Fabio; Masignani, Vega; Pizza, Mariagrazia; Scharenberg, Meike; Schlaeppi, Jean-Marc; Nissum, Mikkel; Liberatori, Sabrina

    2016-01-01

    During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis. PMID:27302108

  5. Immune-Signatures for Lung Cancer Diagnostics: Evaluation of Protein Microarray Data Normalization Strategies

    PubMed Central

    Brezina, Stefanie; Soldo, Regina; Kreuzhuber, Roman; Hofer, Philipp; Gsur, Andrea; Weinhaeusel, Andreas

    2015-01-01

    New minimal invasive diagnostic methods for early detection of lung cancer are urgently needed. It is known that the immune system responds to tumors with production of tumor-autoantibodies. Protein microarrays are a suitable highly multiplexed platform for identification of autoantibody signatures against tumor-associated antigens (TAA). These microarrays can be probed using 0.1 mg immunoglobulin G (IgG), purified from 10 µL of plasma. We used a microarray comprising recombinant proteins derived from 15,417 cDNA clones for the screening of 100 lung cancer samples, including 25 samples of each main histological entity of lung cancer, and 100 controls. Since this number of samples cannot be processed at once, the resulting data showed non-biological variances due to “batch effects”. Our aim was to evaluate quantile normalization, “distance-weighted discrimination” (DWD), and “ComBat” for their effectiveness in data pre-processing for elucidating diagnostic immune-signatures. “ComBat” data adjustment outperformed the other methods and allowed us to identify classifiers for all lung cancer cases versus controls and small-cell, squamous cell, large-cell, and adenocarcinoma of the lung with an accuracy of 85%, 94%, 96%, 92%, and 83% (sensitivity of 0.85, 0.92, 0.96, 0.88, 0.83; specificity of 0.85, 0.96, 0.96, 0.96, 0.83), respectively. These promising data would be the basis for further validation using targeted autoantibody tests.

  6. Nucleolin is a calcium-binding protein.

    PubMed

    Gilchrist, James S C; Abrenica, Bernard; DiMario, Patrick J; Czubryt, Michael P; Pierce, Grant N

    2002-01-01

    We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis. PMID:11948683

  7. Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer

    PubMed Central

    Anderson, Karen S.; Ramachandran, Niroshan; Wong, Jessica; Raphael, Jacob V.; Hainsworth, Eugenie; Demirkan, Gokhan; Cramer, Daniel; Aronzon, Diana; Hodi, F. Stephen; Harris, Lyndsay; Logvinenko, Tanya; LaBaer, Joshua

    2012-01-01

    There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies. PMID:18311903

  8. Multicolor surface plasmon resonance imaging of ink jet-printed protein microarrays.

    PubMed

    Singh, Bipin K; Hillier, Andrew C

    2007-07-15

    We report a technique that utilizes surface plasmon resonance dispersion as a mechanism to provide multicolor contrast for imaging thin molecular films. Illumination of gold surfaces with p-polarized white light in the Kretschmann configuration produces distinct reflected colors due to excitation of surface plasmons and the resulting absorption of specific wavelengths from the source light. In addition, these colors transform in response to the formation of thin molecular films. This process represents a simple detection method for distinguishing between films of varying thickness in sensor applications. As an example, we interrogated a protein microarray formed by a commercial drop-on-demand chemical ink jet printer. Submonolayer films of a test protein (bovine serum albumin) were readily detected by this method. Analysis of the dispersion relations and absorbance sensitivities illustrate the performance and characteristics of this system. Higher detection sensitivity was achieved at angles where red wavelengths coupled to surface plasmons. However, improved contrast and spatial resolution occurred when the angle of incidence was such that shorter wavelengths coupled to the surface plasmons. Simplified optics combined with the robust microarray printing platform are used to demonstrate the applicability of this technique as a rapid and versatile, high-throughput tool for label-free detection of adsorbed films and macromolecules. PMID:17569506

  9. Sterol Carrier Protein-2: Binding Protein for Endocannabinoids

    PubMed Central

    Liedhegner, Elizabeth Sabens; Vogt, Caleb D.; Sem, Daniel S.; Cunningham, Christopher W.

    2015-01-01

    The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs “on-demand,” thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ΔG values of −3.6 and −4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (−6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA. PMID:24510313

  10. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  11. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  12. Production of tissue microarrays, immunohistochemistry staining and digitalization within the human protein atlas.

    PubMed

    Kampf, Caroline; Olsson, Ingmarie; Ryberg, Urban; Sjöstedt, Evelina; Pontén, Fredrik

    2012-01-01

    The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts (1 2). Approximately 250 consecutive sections (4 μm thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (www.proteinatlas.org) (Figure 1) (3 4). The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins

  13. A large-area hemispherical perforated bead microarray for monitoring bead based aptamer and target protein interaction

    PubMed Central

    Choi, Jong Seob; Bae, Sunwoong; Kim, Kyung Hoon; Seo, Tae Seok

    2014-01-01

    Herein, we present a large-area 3D hemispherical perforated microwell structure for a bead based bioassay. Such a unique microstructure enables us to perform the rapid and stable localization of the beads at the single bead level and the facile manipulation of the bead capture and retrieval with high speed and efficiency. The fabrication process mainly consisted of three steps: the convex micropatterned nickel (Ni) mold production from the concave micropatterned silicon (Si) wafer, hot embossing on the polymer matrix to generate the concave micropattened acrylate sheet, and reactive ion etching to make the bottom holes. The large-area hemispherical perforated micropatterned acrylate sheet was sandwiched between two polydimethylsiloxane (PDMS) microchannel layers. The bead solution was injected and recovered in the top PDMS microchannel, while the bottom PDMS microchannel was connected with control lines to exert the hydrodynamic force in order to alter the flow direction of the bead solution for the bead capture and release operation. The streptavidin-coated microbead capture was achieved with almost 100% yield within 1 min, and all the beads were retrieved in 10 s. Lysozyme or thrombin binding aptamer labelled microbeads were trapped on the proposed bead microarray, and the in situ fluorescence signal of the bead array was monitored after aptamer-target protein interaction. The protein-aptamer conjugated microbeads were recovered, and the aptamer was isolated for matrix assisted laser desorption/ionization time-of-flight mass spectrometry analysis to confirm the identity of the aptamer. PMID:25587373

  14. Development of a novel, quantitative protein microarray platform for the multiplexed serological analysis of autoantibodies to cancer-testis antigens.

    PubMed

    Beeton-Kempen, Natasha; Duarte, Jessica; Shoko, Aubrey; Serufuri, Jean-Michel; John, Thomas; Cebon, Jonathan; Blackburn, Jonathan

    2014-10-15

    The cancer-testis antigens are a group of unrelated proteins aberrantly expressed in various cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic vaccines. However, expression levels often vary amongst patients presenting the same cancer type, and these antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of antigen expression may provide correlates of specific cancer types and disease progression. Herein, we describe the development of a novel, readily customizable cancer-testis antigen microarray platform together with robust bioinformatics tools, with which to quantify anti-cancer testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between autoantibodies and tumor antigens, we achieved linearity of response and an autoantibody quantitation limit in the pg/mL range-equating to a million-fold serum dilution. By using oriented attachment of folded, recombinant antigens and a polyethylene glycol microarray surface coating, we attained minimal non-specific antibody binding. Unlike other proteomics methods, which typically use lower affinity interactions between monoclonal antibodies and tumor antigens for detection, the high sensitivity and specificity realized using our autoantibody-based approach may facilitate the development of better cancer biomarkers, as well as potentially enabling pre-symptomatic diagnosis. We illustrated the usage of our platform by monitoring the response of a melanoma patient cohort to an experimental therapeutic NY-ESO-1-based cancer vaccine; inter alia, we found evidence of determinant spreading in individual patients, as well as differential CT antigen expression and epitope usage. PMID:24604332

  15. Tissue microarrays characterise the clinical significance of a VEGF-A protein expression signature in gastrointestinal stromal tumours

    PubMed Central

    Salto-Tellez, M; Nga, M E; Han, H C; Wong, A S-C; Lee, C K; Anuar, D; Ng, S S; Ho, M; Wee, A; Chan, Y H; Soong, R

    2007-01-01

    A tissue microarray analysis of 22 proteins in gastrointestinal stromal tumours (GIST), followed by an unsupervised, hierarchical monothetic cluster statistical analysis of the results, allowed us to detect a vascular endothelial growth factor (VEGF) protein overexpression signature discriminator of prognosis in GIST, and discover novel VEGF-A DNA variants that may have functional significance. PMID:17299397

  16. Binding of transition metals to S100 proteins.

    PubMed

    Gilston, Benjamin A; Skaar, Eric P; Chazin, Walter J

    2016-08-01

    The S100 proteins are a unique class of EF-hand Ca(2+) binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn(2+), Cu(2+) and Mn(2+) ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function. PMID:27430886

  17. PAA: an R/bioconductor package for biomarker discovery with protein microarrays

    PubMed Central

    Turewicz, Michael; Ahrens, Maike; May, Caroline; Marcus, Katrin; Eisenacher, Martin

    2016-01-01

    Summary: The R/Bioconductor package Protein Array Analyzer (PAA) facilitates a flexible analysis of protein microarrays for biomarker discovery (esp., ProtoArrays). It provides a complete data analysis workflow including preprocessing and quality control, uni- and multivariate feature selection as well as several different plots and results tables to outline and evaluate the analysis results. As a main feature, PAA’s multivariate feature selection methods are based on recursive feature elimination (e.g. SVM-recursive feature elimination, SVM-RFE) with stability ensuring strategies such as ensemble feature selection. This enables PAA to detect stable and reliable biomarker candidate panels. Availability and implementation: PAA is freely available (BSD 3-clause license) from http://www.bioconductor.org/packages/PAA/. Contact: michael.turewicz@rub.de or martin.eisenacher@rub.de PMID:26803161

  18. High-throughput identification of putative receptors for cancer-binding peptides using biopanning and microarray analysis

    PubMed Central

    Ferraro, Daniel J; Bhave, Sandeep R; Kotipatruni, Rama P; Hunn, Jeremy C; Wildman, Scott A; Hong, Charles; Dadey, David Y. A.; Muhoro, Lincoln K.; Jaboin, Jerry J; Thotala, Dinesh; Hallahan, Dennis E

    2013-01-01

    Phage-display peptide biopanning has been successfully used to identify cancer-targeting peptides in multiple models. For cancer-binding peptides, identification of the peptide receptor is necessary to demonstrate mechanism of action and to further optimize specificity and target binding. The process of receptor identification can be slow and some peptides may turn out to bind ubiquitous proteins not suitable for further drug development. In this report, we describe a high-throughput method for screening a large number of peptides in parallel to identify peptide receptors, which we have termed “reverse biopanning,” which can then be selected for further development based on their peptide receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically cancers after irradiation. The reverse biopanning process identified 2 peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for the protein tax interacting protein 1 (TIP-1), a protein previously identified in our laboratory to be expressed in the cell surface in tumors and upregulated after exposure to ionizing radiation. We used computational modeling as the initial method for rapid validation of peptide-TIP-1 binding. Pseudo-binding energies were calculated to be −360.645 kcal/mol, −487.239 kcal/mol, and −595.328 kcal/mol for HVGGSSV, TTRYSRV, and NVFFCSV respectively, suggesting that the peptides would have at least similar, if not stronger, binding to TIP-1 compared to the known TIP-1 binding peptide HVGGSSV. We validated peptide in vitro via electrophoretic mobility shift assay, which showed strong binding of RKFLMTTRYSRV and the truncated form TTRYSRV. This method allows for the identification of many peptide receptors and subsequent selection of peptides for further drug development based on the peptide receptor. PMID:23147990

  19. Systematic discovery of Xist RNA binding proteins

    PubMed Central

    Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A.; Bharadwaj, Maheetha; Calabrese, J. Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y.

    2015-01-01

    Summary Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA- protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3′ RNA processing machinery. Xist, an essential lncRNA for X-chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK that participates in Xist-mediated gene silencing and histone modifications, but not Xist localization and Drosophila Split ends homolog Spen that interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. PMID:25843628

  20. Systematic discovery of Xist RNA binding proteins.

    PubMed

    Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A; Bharadwaj, Maheetha; Calabrese, J Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y

    2015-04-01

    Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. PMID:25843628

  1. Cation specific binding with protein surface charges.

    PubMed

    Hess, Berk; van der Vegt, Nico F A

    2009-08-11

    Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of "matching water affinities." This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K(+) < Na(+) < Li(+) of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules. PMID:19666545

  2. Copper-binding protein in Mimulus guttatus

    SciTech Connect

    Robinson, N.J.; Thurman, D.A.

    1985-01-01

    A Cu-binding protein has been purified from the roots of Mimulus guttatus using gel permeation chromatography on Sephadex G-75 and anion exchange chromatography on DEAE Biogel A. The protein has similar properties to putative metallothioneins (MTS) purified from other angiosperms. Putative MT was estimated by measuring the relative percentage incorporation of (/sup 35/S) into fractions containing the protein after HPLC on SW 3000-gel. In the roots of both Cu-tolerant and non tolerant plants synthesis of putative MT is induced by increased Cu concentration in the nutrient solution. The relative percentage incorporation of (/sup 35/S) into putative MT is significantly higher in extracts from the roots of Cu-tolerant than non tolerant M. guttatus after growth in 1 ..mu..M Cu suggesting involvement in the mechanism of tolerance. 22 refs., 2 figs., 1 tab.

  3. DNA and RNA Quadruplex-Binding Proteins

    PubMed Central

    Brázda, Václav; Hároníková, Lucia; Liao, Jack C. C.; Fojta, Miroslav

    2014-01-01

    Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development. PMID:25268620

  4. DNA and RNA quadruplex-binding proteins.

    PubMed

    Brázda, Václav; Hároníková, Lucia; Liao, Jack C C; Fojta, Miroslav

    2014-01-01

    Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development. PMID:25268620

  5. A Crayfish Insulin-like-binding Protein

    PubMed Central

    Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

    2013-01-01

    Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

  6. Identification of DNA-binding and protein-binding proteins using enhanced graph wavelet features.

    PubMed

    Zhu, Yuan; Zhou, Weiqiang; Dai, Dao-Qing; Yan, Hong

    2013-01-01

    Interactions between biomolecules play an essential role in various biological processes. For predicting DNA-binding or protein-binding proteins, many machine-learning-based techniques have used various types of features to represent the interface of the complexes, but they only deal with the properties of a single atom in the interface and do not take into account the information of neighborhood atoms directly. This paper proposes a new feature representation method for biomolecular interfaces based on the theory of graph wavelet. The enhanced graph wavelet features (EGWF) provides an effective way to characterize interface feature through adding physicochemical features and exploiting a graph wavelet formulation. Particularly, graph wavelet condenses the information around the center atom, and thus enhances the discrimination of features of biomolecule binding proteins in the feature space. Experiment results show that EGWF performs effectively for predicting DNA-binding and protein-binding proteins in terms of Matthew's correlation coefficient (MCC) score and the area value under the receiver operating characteristic curve (AUC). PMID:24334394

  7. Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

    PubMed

    Bal, Gürkan; Futschik, Matthias E; Hartl, Daniela; Ringel, Frauke; Kamhieh-Milz, Julian; Sterzer, Viktor; Hoheisel, Jörg D; Alhamdani, Mohamed S S; Salama, Abdulgabar

    2016-02-01

    The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour-suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti-nuclear autoantibodies in a subset of ITP patients: anti-PCNA, anti-SmD, anti-Ro/SSA60, anti-Ro/SSA52, anti-La/SSB and anti-RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration. PMID:26628061

  8. Competitive protein binding assay for piritrexim

    SciTech Connect

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. )

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  9. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis.

    PubMed

    Kempsell, Karen E; Kidd, Stephen P; Lewandowski, Kuiama; Elmore, Michael J; Charlton, Sue; Yeates, Annemarie; Cuthbertson, Hannah; Hallis, Bassam; Altmann, Daniel M; Rogers, Mitch; Wattiau, Pierre; Ingram, Rebecca J; Brooks, Tim; Vipond, Richard

    2015-01-01

    A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis "infectome." These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from the

  10. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

    PubMed Central

    Kempsell, Karen E.; Kidd, Stephen P.; Lewandowski, Kuiama; Elmore, Michael J.; Charlton, Sue; Yeates, Annemarie; Cuthbertson, Hannah; Hallis, Bassam; Altmann, Daniel M.; Rogers, Mitch; Wattiau, Pierre; Ingram, Rebecca J.; Brooks, Tim; Vipond, Richard

    2015-01-01

    A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis “infectome.” These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from

  11. Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic

    PubMed Central

    Zhang, Hai-nan; Yang, Lina; Ling, Jian-ya; Czajkowsky, Daniel M.; Wang, Jing-Fang; Zhang, Xiao-Wei; Zhou, Yi-Ming; Ge, Feng; Yang, Ming-kun; Xiong, Qian; Guo, Shu-Juan; Le, Huang-Ying; Wu, Song-Fang; Yan, Wei; Liu, Bingya; Zhu, Heng; Chen, Zhu; Tao, Sheng-ce

    2015-01-01

    Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies. PMID:26598702

  12. Landscape of protein-small ligand binding modes.

    PubMed

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  13. Regulation of Pluripotency by RNA Binding Proteins

    PubMed Central

    Ye, Julia; Blelloch, Robert

    2015-01-01

    Establishment, maintenance, and exit from pluripotency require precise coordination of a cell’s molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells. PMID:25192462

  14. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  15. Computational Design of DNA-Binding Proteins.

    PubMed

    Thyme, Summer; Song, Yifan

    2016-01-01

    Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering. PMID:27094297

  16. Plastic protein microarray to investigate the molecular pathways of magnetic nanoparticle-induced nanotoxicity

    NASA Astrophysics Data System (ADS)

    Liu, Yingshuai; Li, Xuelian; Bao, Shujuan; Lu, Zhisong; Li, Qing; Li, Chang Ming

    2013-05-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) (about 15 nm) were synthesized via a hydrothermal method and characterized by field emission scanning electron microscopy, transmission electron microscopy, dynamic light scattering, x-ray diffraction, and vibrating sample magnetometer. The molecular pathways of SPIONs-induced nanotoxicity was further investigated by protein microarrays on a plastic substrate from evaluation of cell viability, reactive oxygen species (ROS) generation and cell apoptosis. The experimental results reveal that 50 μg ml-1 or higher levels of SPIONs cause significant loss of cell viability, considerable generation of ROS and cell apoptosis. It is proposed that high level SPIONs could induce cell apoptosis via a mitochondria-mediated intrinsic pathway by activation of caspase 9 and caspase 3, an increase of the Bax/Bcl-2 ratio, and down-regulation of HSP70 and HSP90 survivor factors.

  17. Printing of protein microarrays via a capillary-free fluid jetting mechanism.

    PubMed

    Barron, J A; Young, H D; Dlott, D D; Darfler, M M; Krizman, D B; Ringeisen, B R

    2005-11-01

    Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microm and approximately 500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer. PMID:16196096

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

    ERIC Educational Resources Information Center

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

    2010-01-01

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

  19. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    NASA Astrophysics Data System (ADS)

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  20. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    PubMed

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. PMID:26805756

  1. Ligand configurational entropy and protein binding.

    PubMed

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

    2007-01-30

    The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing approximately 25 kcal/mol (4.184 kJ/kcal) to DeltaG degrees. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms. The results highlight the potential to gain affinity by designing conformationally restricted ligands and have implications for the formulation of energy models for ligand scoring. PMID:17242351

  2. Ligand configurational entropy and protein binding

    PubMed Central

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

    2007-01-01

    The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing ∼25 kcal/mol (4.184 kJ/kcal) to ΔG°. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms. The results highlight the potential to gain affinity by designing conformationally restricted ligands and have implications for the formulation of energy models for ligand scoring. PMID:17242351

  3. Alternative polyadenylation and RNA-binding proteins.

    PubMed

    Erson-Bensan, Ayse Elif

    2016-08-01

    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  4. Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach

    PubMed Central

    Lessa-Aquino, Carolina; Borges Rodrigues, Camila; Pablo, Jozelyn; Sasaki, Rie; Jasinskas, Algis; Liang, Li; Wunder, Elsio A.; Ribeiro, Guilherme S.; Vigil, Adam; Galler, Ricardo; Molina, Douglas; Liang, Xiaowu; Reis, Mitermayer G.; Ko, Albert I.; Medeiros, Marco Alberto; Felgner, Philip L.

    2013-01-01

    Background Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease. Methodology In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results. Principal findings We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients. Conclusions Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for

  5. The modular architecture of protein-protein binding interfaces.

    PubMed

    Reichmann, D; Rahat, O; Albeck, S; Meged, R; Dym, O; Schreiber, G

    2005-01-01

    Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. PMID:15618400

  6. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  7. Prednisolone protein binding in renal transplant patients.

    PubMed Central

    Reece, P A; Disney, A P; Stafford, I; Shastry, J C

    1985-01-01

    Prednisolone pharmacokinetics and protein binding characteristics were studied in 10 renal transplant patients with various degrees of renal function (serum creatinine: 80-380 mumol/l) who received their usual oral maintenance dose of prednisolone (0.18 +/- 0.04 mg/kg). Plasma was assayed for prednisolone and hydrocortisone by h.p.l.c. and free prednisolone concentrations were determined in each sample by a rapid ultrafiltration technique. Free prednisolone area under curve (AUCu) ranged from 101 to 436 ng ml-1 h and was 6.3 to 15.0% of total prednisolone AUC. The fraction AUCu/AUC was closely related to serum albumin and creatinine concentrations determined at the time of study (multilinear regression correlation coefficient r2 = 0.830, P less than 0.0001); elevated serum creatinine and low albumin concentrations were associated with a higher % free. These results suggest that much of the variability in prednisolone protein binding could be attributed to inter-patient variability in serum albumin and creatinine concentrations. Total prednisolone concentrations would be potentially misleading in any comparisons made between patient groups with different renal function. PMID:3899153

  8. ASSESSMENT OF THE SWINE PROTEIN-ANNOTATED OLIGONUCLEOTIDE MICROARRAY AND UTILITY OF THE ARRAYS FOR EQTL AND TRANSCRIPTIONAL PROFILING STUDIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), Bronchial lymph node (BLN) and Lung. Four LD samples were used to assess the stringency of hybridization conditions com...

  9. Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as antibody labels to increase the fluorescence signal and sensitivity. Ep...

  10. Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins.

    PubMed

    Shimizu, M; Yoshida, T; Toda, T; Iwashima, A; Mitsunaga, T

    1996-01-01

    A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, Kd and Bmax values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein. PMID:27299548

  11. Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach

    PubMed Central

    2014-01-01

    Background Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery. Methods A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. One antigen was further characterised and its sequence and structure were analysed in silico. Real-time polymerase chain reaction was used to analyse transcript expression throughout development, and immunoblotting and enzyme-linked immunosorbent assays employed to determine antigen recognition by antibody samples. Results Several known and novel antigens were discovered, two of which showed up-regulated transcription in schistosomula. One novel antigen, termed S. japonicum Ly-6-like protein 1 (Sj-L6L-1), was further characterised and shown to share structural and sequence features with the Ly-6 protein family. It was found to be present in the worm tegument and expressed in both the larval and adult worms, but was found to be antigenic only in the lungs that the larvae migrate to and traverse. Conclusions This study represents a novel approach to vaccine antigen discovery and may contribute to schistosome vaccine development against this important group of human and veterinary pathogens. PMID:24964958

  12. Microcontact printing trapping air: A versatile tool for protein microarray fabrication

    NASA Astrophysics Data System (ADS)

    Filipponi, Luisa; Nicolau, Dan V.

    2006-01-01

    The present work introduces a new method for the fabrication of protein micro-patterns, microcontact printing trapping air. The method is based on microcontact printing, a well-established soft-lithographic technique for printing bioactive protein patterns. Usually, the stamp used is made of poly(dimethylsiloxane) obtained by replicating a lithographically microfabricated silicon master. In microcontact printing, the dimensions of the features in the stamp are critical, since the high compressibility of poly(dimethylsiloxane) causes high aspect ratio features to collapse, leading to the printing of undesired areas. In most cases, this is an unwanted effect, which interferes with the printing quality. In this work we used a poly(dimethylsiloxane)stamp bearing an array of micro-posts which, when placed over a flat surface, collapses with consequent formation of an air gap around the entire array. This effect is linked to the distance between the posts that form the array and can be exploited for the fabrication of protein microarrays having a remarkably low background noise for fluorescence detection.

  13. A Protein Microarray Signature of Autoantibody Biomarkers for the Early Detection of Breast Cancer

    PubMed Central

    Anderson, Karen S.; Sibani, Sahar; Wallstrom, Garrick; Qiu, Ji; Mendoza, Eliseo A.; Raphael, Jacob; Hainsworth, Eugenie; Montor, Wagner R.; Wong, Jessica; Park, Jin G.; Lokko, Naa; Logvinenko, Tanya; Ramachandran, Niroshan; Godwin, Andrew K.; Marks, Jeffrey; Engstrom, Paul; LaBaer, Joshua

    2011-01-01

    Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins.. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4,988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a pre-screen was performed to eliminate uninformative antigens. Sera from stage I–III IBC (n=53) and healthy women (n=53) were screened for AAb to all 4,988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p<0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p<0.05). These 761 antigens were screened using an independent set of IBC sera (n=51) and sera from women with benign breast disease (BBD) (n=39). From these, 119 antigens had a partial area under the ROC curve (p<0.05), with sensitivities ranging from 9–40% at >91% specificity. 28 of these antigens were confirmed using an independent serum cohort (n=51 cases/38 controls, p<0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC=0.756). These are potential biomarkers for the early detection of breast cancer. PMID:20977275

  14. STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition

    PubMed Central

    Bonham, Andrew J.; Wenta, Nikola; Osslund, Leah M.; Prussin, Aaron J.; Vinkemeier, Uwe; Reich, Norbert O.

    2013-01-01

    The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design. PMID:23180800

  15. Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E.

    PubMed

    Renault, N K; Gaddipati, S R; Wulfert, F; Falcone, F H; Mirotti, L; Tighe, P J; Wright, V; Alcocer, M J C

    2011-02-01

    Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described

  16. Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein.

    PubMed

    Sawicka, Kirsty; Bushell, Martin; Spriggs, Keith A; Willis, Anne E

    2008-08-01

    PTB (polypyrimidine-tract-binding protein) is a ubiquitous RNA-binding protein. It was originally identified as a protein with a role in splicing but it is now known to function in a large number of diverse cellular processes including polyadenylation, mRNA stability and translation initiation. Specificity of PTB function is achieved by a combination of changes in the cellular localization of this protein (its ability to shuttle from the nucleus to the cytoplasm is tightly controlled) and its interaction with additional proteins. These differences in location and trans-acting factor requirements account for the fact that PTB acts both as a suppressor of splicing and an activator of translation. In the latter case, the role of PTB in translation has been studied extensively and it appears that this protein is required for an alternative form of translation initiation that is mediated by a large RNA structural element termed an IRES (internal ribosome entry site) that allows the synthesis of picornaviral proteins and cellular proteins that function to control cell growth and cell death. In the present review, we discuss how PTB regulates these disparate processes. PMID:18631133

  17. Structural neighboring property for identifying protein-protein binding sites

    PubMed Central

    2015-01-01

    Background The protein-protein interaction plays a key role in the control of many biological functions, such as drug design and functional analysis. Determination of binding sites is widely applied in molecular biology research. Therefore, many efficient methods have been developed for identifying binding sites. In this paper, we calculate structural neighboring property through Voronoi diagram. Using 6,438 complexes, we study local biases of structural neighboring property on interface. Results We propose a novel statistical method to extract interacting residues, and interacting patches can be clustered as predicted interface residues. In addition, structural neighboring property can be adopted to construct a new energy function, for evaluating docking solutions. It includes new statistical property as well as existing energy items. Comparing to existing methods, our approach improves overall Fnat value by at least 3%. On Benchmark v4.0, our method has average Irmsd value of 3.31Å and overall Fnat value of 63%, which improves upon Irmsd of 3.89 Å and Fnat of 49% for ZRANK, and Irmsd of 3.99Å and Fnat of 46% for ClusPro. On the CAPRI targets, our method has average Irmsd value of 3.46 Å and overall Fnat value of 45%, which improves upon Irmsd of 4.18 Å and Fnat of 40% for ZRANK, and Irmsd of 5.12 Å and Fnat of 32% for ClusPro. Conclusions Experiments show that our method achieves better results than some state-of-the-art methods for identifying protein-protein binding sites, with the prediction quality improved in terms of CAPRI evaluation criteria. PMID:26356630

  18. RNA Bind-n-Seq: Measuring the Binding Affinity Landscape of RNA-Binding Proteins.

    PubMed

    Lambert, Nicole J; Robertson, Alex D; Burge, Christopher B

    2015-01-01

    RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function of these factors will require the identification of each RBP's distinct RNA binding specificity. RNA Bind-n-Seq (RBNS) is a high-throughput, cost-effective in vitro method capable of resolving sequence and secondary structure preferences of RBPs. Dissociation constants can also be inferred from RBNS data when provided with additional experimental information. Here, we describe the experimental procedures to perform RBNS and discuss important parameters of the method and ways that the experiment can be tailored to the specific RBP under study. Additionally, we present the conceptual framework and execution of the freely available RBNS computational pipeline and describe the outputs of the pipeline. Different approaches to quantify binding specificity, quality control metrics, and estimation of binding constants are also covered. PMID:26068750

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

    PubMed Central

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

    2016-01-01

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

  20. Glycan Masking of Plasmodium vivax Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

    PubMed Central

    Janes, Joel; Gurumoorthy, Sairam; Gibson, Claire; Melcher, Martin; Chitnis, Chetan E.; Wang, Ruobing; Schief, William R.; Smith, Joseph D.

    2013-01-01

    Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development. PMID:23853575

  1. Identification and isoprenylation of plant GTP-binding proteins.

    PubMed

    Biermann, B; Randall, S K; Crowell, D N

    1996-08-01

    To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies). PMID:8843944

  2. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations.

    PubMed

    Delalat, Bahman; Rojas-Canales, Darling M; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J; Penko, Daniella; Drogemuller, Christopher J; Loudovaris, Thomas; Coates, Patrick T H; Voelcker, Nicolas H

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  3. Protein Function Annotation By Local Binding Site Surface Similarity

    PubMed Central

    Spitzer, Russell; Cleves, Ann E.; Varela, Rocco; Jain, Ajay N.

    2013-01-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against approximately 60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that pre-dated query protein biochemical annotation for five out of the eight query proteins. A panel of twelve currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins. PMID:24166661

  4. RNA-binding protein nucleolin in disease.

    PubMed

    Abdelmohsen, Kotb; Gorospe, Myriam

    2012-06-01

    Nucleolin is a multifunctional protein localized primarily in the nucleolus, but also found in the nucleoplasm, cytoplasm and cell membrane. It is involved in several aspects of DNA metabolism, and participates extensively in RNA regulatory mechanisms, including transcription, ribosome assembly, mRNA stability and translation, and microRNA processing. Nucleolin's implication in disease is linked to its ability to associate with target RNAs via its four RNA-binding domains and its arginine/glycin-rich domain. By modulating the post-transcriptional fate of target mRNAs, which typically bear AU-rich and/or G-rich elements, nucleolin has been linked to cellular events that influence disease, notably cell proliferation and protection against apoptotic death. Through its diverse RNA functions, nucleolin is increasingly implicated in pathological processes, particularly cancer and viral infection. Here, we review the RNA-binding activities of nucleolin, its influence on gene expression patterns, and its impact upon diseases. We also discuss the rising interest in targeting nucleolin therapeutically. PMID:22617883

  5. RNA-binding protein nucleolin in disease

    PubMed Central

    Abdelmohsen, Kotb; Gorospe, Myriam

    2012-01-01

    Nucleolin is a multifunctional protein localized primarily in the nucleolus, but also found in the nucleoplasm, cytoplasm and cell membrane. It is involved in several aspects of DNA metabolism, and participates extensively in RNA regulatory mechanisms, including transcription, ribosome assembly, mRNA stability and translation, and microRNA processing. Nucleolin’s implication in disease is linked to its ability to associate with target RNAs via its four RNA-binding domains and its arginine/glycin-rich domain. By modulating the post-transcriptional fate of target mRNAs, which typically bear AU-rich and/or G-rich elements, nucleolin has been linked to cellular events that influence disease, notably cell proliferation and protection against apoptotic death. Through its diverse RNA functions, nucleolin is increasingly implicated in pathological processes, particularly cancer and viral infection. Here, we review the RNA-binding activities of nucleolin, its influence on gene expression patterns, and its impact upon diseases. We also discuss the rising interest in targeting nucleolin therapeutically. PMID:22617883

  6. Latent TGF-β-binding proteins

    PubMed Central

    Robertson, Ian B.; Horiguchi, Masahito; Zilberberg, Lior; Dabovic, Branka; Hadjiolova, Krassimira; Rifkin, Daniel B.

    2016-01-01

    The LTBPs (or latent transforming growth factor β binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFβ precursor in the trans golgi network but LAP and TGFβ remain strongly bound through non-covalent interactions. LAP, TGFβ, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFβ latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFβ activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFβ regulation, TGFβ-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly. PMID:25960419

  7. Characterizing the morphology of protein binding patches.

    PubMed

    Malod-Dognin, Noël; Bansal, Achin; Cazals, Frédéric

    2012-12-01

    Let the patch of a partner in a protein complex be the collection of atoms accounting for the interaction. To improve our understanding of the structure-function relationship, we present a patch model decoupling the topological and geometric properties. While the geometry is classically encoded by the atomic positions, the topology is recorded in a graph encoding the relative position of concentric shells partitioning the interface atoms. The topological-geometric duality provides the basis of a generic dynamic programming-based algorithm comparing patches at the shell level, which may favor topological or geometric features. On the biological side, we address four questions, using 249 cocrystallized heterodimers organized in biological families. First, we dissect the morphology of binding patches and show that Nature enjoyed the topological and geometric degrees of freedom independently while retaining a finite set of qualitatively distinct topological signatures. Second, we argue that our shell-based comparison is effective to perform atomic-level comparisons and show that topological similarity is a less stringent than geometric similarity. We also use the topological versus geometric duality to exhibit topo-rigid patches, whose topology (but not geometry) remains stable upon docking. Third, we use our comparison algorithms to infer specificity-related information amidst a database of complexes. Finally, we exhibit a descriptor outperforming its contenders to predict the binding affinities of the affinity benchmark. The softwares developed with this article are availablefrom http://team.inria.fr/abs/vorpatch_compatch/. PMID:22806945

  8. Cellular retinol-binding protein and retinoic acid-binding protein in rat testes: effect of retinol depletion.

    PubMed

    Ong, D E; Tsai, C H; Chytil, F

    1976-02-01

    Testes of rats contain two cellular binding proteins of interest in vitamin A metabolism. One protein binds retinoic acid with high specificity; the other binds retinol with high specificity. When the cellular retinol-binding protein was partially purified from rat testes, it exhibited fluorescence excitation and emission spectra similar to that of all-trans-retinol in hexane. Exposure of this preparation to UV light destroyed this fluorescence but spectra identical to the original were obtained after addition of retinol. Hexane extracts of the binding protein had fluorescence spectra identical to all-trans-retinol, suggesting that this compound is bound to the protein in vivo. Extracts of testes from retinol depleted rats were submitted to gel filtration but failed to show a retinol-like fluorescence at the elution position of retinol binding protein. This fluorescence was observed in the preparations from pair fed control animals. However, after addition of all-trans-retinol to the extracts from the depleted rats, fluorescence at that elution position was observed. This indicates that in testes of retinol depleted rats the cellular retinol binding protein is present but without bound retinol, in contrast to the non-depleted rats where 30-43% of the binding protein had bound retinol. The amounts of cellular retinol binding protein and retinoic acid binding protein in testes, as determined by sucrose gradient centrifugation, were found to be similar for retinol depleted and pair fed control rats. PMID:942996

  9. How Does Confinement Change Ligand-Receptor Binding Equilibrium? Protein Binding in Nanopores and Nanochannels.

    PubMed

    Tagliazucchi, Mario; Szleifer, Igal

    2015-10-01

    We present systematic studies for the binding of small model proteins to ligands attached to the inner walls of long nanochannels and short nanopores by polymeric tethers. Binding of proteins to specific ligands inside nanometric channels and pores leads to changes in their ionic conductance, which have been exploited in sensors that quantify the concentration of the proteins in solution. The theoretical predictions presented in this work are aimed to provide a fundamental understanding of protein binding under geometrically confined environments and to guide the design of this kind of nanochannel-based sensors. The theory predicts that the fraction of the channel volume filled by bound proteins is a nonmonotonic function of the channel radius, the length of the tethers, the surface density of the ligands and the size of the proteins. Notably, increasing the density of ligands, decreasing the size of the channel or increasing the size of the protein may lead to a decrease of the fraction of the channel volume filled by bound proteins. These results are explained from the incomplete binding of proteins to the ligands due to repulsive protein-protein and protein-ligand steric interactions. Our work suggests strategies to optimize the change in conductance due to protein binding, for example: (i) proteins much smaller than the radius of the channel may effectively block the channel if tethers of appropriate length are used, and (ii) a large decrease in conductance upon protein binding can be achieved if the channel and the protein are oppositely charged. PMID:26368839

  10. Detection of secondary binding sites in proteins using fragment screening

    PubMed Central

    Ludlow, R. Frederick; Verdonk, Marcel L.; Saini, Harpreet K.; Tickle, Ian J.; Jhoti, Harren

    2015-01-01

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets. PMID:26655740

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

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

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

  12. Minimalistic predictor of protein binding energy: contribution of solvation factor to protein binding.

    PubMed

    Choi, Jeong-Mo; Serohijos, Adrian W R; Murphy, Sean; Lucarelli, Dennis; Lofranco, Leo L; Feldman, Andrew; Shakhnovich, Eugene I

    2015-02-17

    It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software. PMID:25692584

  13. Photoaffinity labeling of retinoic acid-binding proteins.

    PubMed Central

    Bernstein, P S; Choi, S Y; Ho, Y C; Rando, R R

    1995-01-01

    Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7846032

  14. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

    PubMed

    Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  15. Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

    PubMed

    Palacín, Arantxa; Gómez-Casado, Cristina; Rivas, Luis A; Aguirre, Jacobo; Tordesillas, Leticia; Bartra, Joan; Blanco, Carlos; Carrillo, Teresa; Cuesta-Herranz, Javier; de Frutos, Consolación; Alvarez-Eire, Genoveva García; Fernández, Francisco J; Gamboa, Pedro; Muñoz, Rosa; Sánchez-Monge, Rosa; Sirvent, Sofía; Torres, María J; Varela-Losada, Susana; Rodríguez, Rosalía; Parro, Victor; Blanca, Miguel; Salcedo, Gabriel; Díaz-Perales, Araceli

    2012-01-01

    The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens. PMID:23272072

  16. DNA Shape versus Sequence Variations in the Protein Binding Process.

    PubMed

    Chen, Chuanying; Pettitt, B Montgomery

    2016-02-01

    The binding process of a protein with a DNA involves three stages: approach, encounter, and association. It has been known that the complexation of protein and DNA involves mutual conformational changes, especially for a specific sequence association. However, it is still unclear how the conformation and the information in the DNA sequences affects the binding process. What is the extent to which the DNA structure adopted in the complex is induced by protein binding, or is instead intrinsic to the DNA sequence? In this study, we used the multiscale simulation method to explore the binding process of a protein with DNA in terms of DNA sequence, conformation, and interactions. We found that in the approach stage the protein can bind both the major and minor groove of the DNA, but uses different features to locate the binding site. The intrinsic conformational properties of the DNA play a significant role in this binding stage. By comparing the specific DNA with the nonspecific in unbound, intermediate, and associated states, we found that for a specific DNA sequence, ∼40% of the bending in the association forms is intrinsic and that ∼60% is induced by the protein. The protein does not induce appreciable bending of nonspecific DNA. In addition, we proposed that the DNA shape variations induced by protein binding are required in the early stage of the binding process, so that the protein is able to approach, encounter, and form an intermediate at the correct site on DNA. PMID:26840719

  17. Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

    PubMed Central

    Zhou, Jerry; Belov, Larissa; Solomon, Michael J.; Chan, Charles; Clarke, Stephen J.; Christopherson, Richard I.

    2011-01-01

    The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1. A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs). The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single ‘marker’ has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC

  18. Glycan microarrays for decoding the glycome

    PubMed Central

    Rillahan, Cory D.; Paulson, James C.

    2011-01-01

    In the last decade glycan microarrays have revolutionized the analysis of the specificity of glycan binding proteins, providing information that simultaneously illuminates the biology mediated by them and decodes the information content of the glycome. Numerous methods have emerged for arraying glycans in a ‘chip’ format, and glycan libraries have been assembled that address the diversity of the human glycome. Such arrays have been successfully used for analysis of glycan binding proteins that mediate mammalian biology, host-pathogen interactions, immune recognition of glycans relevant to vaccine production and cancer antigens. This review covers the development of glycan microarrays and applications that have provided insights into the roles of mammalian and microbial glycan binding proteins. PMID:21469953

  19. Heterochromatin Protein 1 Binding Protein 3 Expression as a Candidate Marker of Intrinsic 5-Fluorouracil Resistance

    PubMed Central

    HADAC, JAMIE N.; MILLER, DEVON D.; GRIMES, IAN C.; CLIPSON, LINDA; NEWTON, MICHAEL A.; SCHELMAN, WILLIAM R.; HALBERG, RICHARD B.

    2016-01-01

    Background Despite receiving post-operative 5-fluorouracil (5-FU)-based chemotherapy, approximately 50% of patients with stage IIIC colon cancer experience recurrence. Currently, no molecular signature can predict response to 5-FU. Materials and Methods Mouse models of colon cancer have been developed and characterized. Individual tumors in these mice can be longitudinally monitored and assessed to identify differences between those that are responsive and those that are resistant to therapy. Gene expression was analyzed in serial biopsies that were collected before and after treatment with 5-FU. Colon tumors had heterogeneous responses to treatment with 5-FU. Microarray analysis of pretreatment biopsies revealed that Hp1bp3, a gene encoding heterochromatin protein 1 binding protein 3, was differentially expressed between sensitive and resistant tumors. Conclusion Using mouse models of human colorectal cancer, Hp1bp3 was identified as a candidate marker of intrinsic 5-FU resistance and may represent a potential biomarker for patient stratification or a target of clinical importance. PMID:26976970

  20. Laser trapping and patterning of protein microcrystals: Toward highly integrated protein microarrays

    NASA Astrophysics Data System (ADS)

    Hosokawa, Yoichiroh; Matsumura, Satoshi; Masuhara, Hiroshi; Ikeda, Keiko; Shimo-oka, Ai; Mori, Hajime

    2004-09-01

    Some insect virus infections occlude into a crystalline matrix consisting of a protein named polyhedrin. The shape of the matrix is a cubic polyhedron of the size of a few micrometers. Recently it was shown that these polyhedra could immobilize various functional proteins within them. Therefore, the polyhedron is interesting as an element in a protein chip. In this work, individual polyhedra were arrayed and bonded under a microscope by focused laser beams, with the aim of fabricating a highly integrated protein chip. The polyhedron was trapped and transferred to a suitable position on a polymer substrate by optical trapping with a 1064nmNd3+:YAG (YAG, yttrium aluminum garnet) laser. To bond the polyhedron on the substrate, the polymer surface was mechanically and chemically modified by multiphoton absorption of a 120fs, 800nm femtosecond Ti: sapphire laser, which results in strong adhesion of the polyhedron to the substrate. The arraying and bonding of polyhedra were successful, to a precision of about 1μm, with this procedure. The biological activity of polyhedra after these laser irradiations was confirmed by the fluorescence of green fluorescent protein occluded in the polyhedrin matrix.

  1. Partial characterization of GTP-binding proteins in Neurospora

    SciTech Connect

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-08-14

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. (/sup 35/S)GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of (/sup 35/S)GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin.

  2. Dot-blot assay for heparin-binding proteins

    SciTech Connect

    Hirose, N.; Krivanek, M.; Jackson, R.L.; Cardin, A.D.

    1986-08-01

    A method for the detection and quantitation of picomole amounts of heparin-binding proteins is described. Proteins are first spotted on nitrocellulose and then incubated with /sup 125/I-heparin. Binding of heparin to the proteins is detected by radioautography and quantitated by scanning densitometry; proteins are quantitated by densitometric analysis of the amido black stained nitrocellulose. Heparin-binding was time-dependent and sensitive to the presence of metal ions, urea, and detergents (anionic, nonionic, and zwitterionic). The divalent cations Ca/sup 2 +/ and Mg/sup 2 +/ and the zwitterionic detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate increased heparin binding whereas NaCl, urea, sodium dodecylsulfate, and La3+ decreased binding. This assay is applicable to the identification and characterization of a variety of heparin-binding proteins.

  3. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  4. Inkjet printing of protein microarrays on freestanding polymeric nanofilms for spatio-selective cell culture environment.

    PubMed

    Fujie, Toshinori; Desii, Andrea; Ventrelli, Letizia; Mazzolai, Barbara; Mattoli, Virgilio

    2012-12-01

    In the last years, an increasing interest in bio-hybrid systems for what concerns the precise control of cell-material interactions has emerged. This trend leads towards the development of new nano-structured devices such as bioMEMS, tissue-engineering scaffolds, biosensors, etc. In the present study, we focused on the development of a spatio-selective cell culture environment based on the inkjet printing of bio-patterns on polymeric ultra-thin films (nanofilms) composed of poly(methylmethacrylate) (PMMA). Freestanding PMMA nanofilms having hundreds-of-nm thickness were prepared by spin-coating. Different shapes of cell adhesion promoters such as poly (L-lysine) (PLL) were micropatterned by inkjet printing. Moreover, to promote cell adhesion, the surface of PLL microarrays was modified with fibronectin via electorostatic interaction. The selective deposition of C2C12 skeletal muscle cells was confirmed and their viability was qualitatively assessed after 24 h. The combination of muscular cells with protein micropatterned freestanding nanofilm is beneficial for the implementation of new bio-hybrid system in muscular tissue engineering. PMID:22986760

  5. Carbohydrate microarray for the detection of glycan-protein interactions using metal-enhanced fluorescence.

    PubMed

    Yang, Jie; Moraillon, Anne; Siriwardena, Aloysius; Boukherroub, Rabah; Ozanam, François; Gouget-Laemmel, Anne Chantal; Szunerits, Sabine

    2015-04-01

    Carbohydrate arrays are potentially one of the most attractive tools to study carbohydrate-based interactions. This paper describes a new analytical platform that exploits metal-enhanced fluorescence for the sensitive and selective screening of carbohydrate-lectin interactions. The chip consists of a glass slide covered with gold nanostructures, postcoated with a thin layer of amorphous silicon-carbon alloy (a-Si0.8C0.2:H). An immobilization strategy based on the formation of a covalent bond between propargyl-terminated glycans and surface-linked azide groups was used to attach various glycans at varying surface densities onto the interface and to fabricate a carbohydrate array via efficient local "click" chemistry strategy. The specific association of the new interface with fluorescently labeled lectins was assessed by fluorescence imaging and an excellent selectivity to specific proteins was achieved. Optimization of the surface architecture and the plasmonic transducer resulted in an enhancement of the fluorescence intensity by 1 order of magnitude, when compared to the corresponding substrate devoid of gold nanostructures. The limit of detection (LOD) of such microarrays is in the picomolar range, making it a promising system for development in pharmaceutical or biomedical applications. PMID:25729928

  6. Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays.

    PubMed

    Le Goff, Gaelle C; Desmet, Cloé; Brès, Jean-Charles; Rigal, Dominique; Blum, Loïc J; Marquette, Christophe A

    2010-12-15

    We are reporting here a low cost colorimetric device for high-throughput multiplexed blood group genotyping and allergy diagnosis, displayed as an automated 96-well microtiter plate format. A porous polymeric membrane sealed at the bottom of each well accounts for the sensor support. For each sensing unit, a 6×6 matrix of specific probes is spotted on the external surface of the membrane resulting in 5 mm(2) microarrays. Thanks to the membrane porosity, reagents dispensed into the well can be eliminated through vacuum soaking. This unusual design drastically reduces the assay background signal. The system was first validated on robust models composed of either two complementary oligonucleotide sequences or one allergen/specific rabbit IgG pair. The quality of both oligonucleotide and protein immobilisation on the membrane substrate was then demonstrated together with the capacity to use the arrayed biomolecules as probes for the quantitative detection of specific targets (respectively complementary oligonucleotide and specific antibody). On the basis of these good results, two multiplex assays were developed for crude biological samples testing, focussing on two human in vitro diagnosis applications: a hybridisation assay for multiplex blood group genotyping and a multiparametric immunoassay for allergy diagnosis. In both cases, the transfer to crude biological samples testing was successful i.e. high signal to noise ratio of the stained membranes, reproducibility and good correlation with results obtained using routine testing procedures. PMID:20663657

  7. Detection of hybridization of protein microarrays using an oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Lu, Heng; Wen, Juan; Wang, Xu; Yuan, Kun; Lu, Huibin; Zhou, Yueliang; Jin, Kui-Juan; Yang, Guozhen; Li, Wei; Ruan, Kangcheng

    2010-07-01

    Mouse-Immunoglobulin G (mouse-IgG) with different concentrations in a range from 1000 to 0.0128 μg/mL and a specific hybridization with goat anti-mouse IgG were detected successfully by using an oblique-incidence reflectivity difference (OI-RD) method. Two detection signals, consisting of an imaginary part (Im{Δp-Δs}) and a real part (Re{Δp-Δs}) of OI-RD, were obtained simultaneously. The detection results of hybridization by OI-RD were in accord with that of traditional fluorescent scans. In particular, we label-freely detected the washed mouse-IgG microarray with a series of concentrations and acquired a linear correlation between OI-RD intensities and the protein concentrations in logarithmic coordinates. The detection sensitivity of OI-RD can reach 14 fg. These experimental results suggest that the OI-RD method has potential applications in proteomics and clinical diagnosis.

  8. Alteration and localization of glycan-binding proteins in human hepatic stellate cells during liver fibrosis.

    PubMed

    Zhong, Yaogang; Qin, Yannan; Dang, Liuyi; Jia, Liyuan; Zhang, Zhiwei; Wu, Haoxiang; Cui, Jihong; Bian, Huijie; Li, Zheng

    2015-10-01

    Glycan-binding proteins (GBPs) play an important role in cell adhesion, bacterial/viral infection, and cellular signaling pathways. However, little is known about the precision alteration of GBPs referred to pathological changes in hepatic stellate cells (HSCs) during liver fibrosis. Here, the carbohydrate microarrays were used to probe the alteration of GBPs in the activated HSCs and quiescent HSCs. As a result, 12 carbohydrates (e.g. Gal, GalNAc, and Man-9Glycan) showed increased signal, while seven carbohydrates (e.g. NeuAc, Lac, and GlcNAc-O-Ser) showed decreased signal in activated HSCs. Three carbohydrates (Gal, GalNAc, and NeuAc) were selected and subsequently used to validate the results of the carbohydrate microarrays as well as assess the distribution and localization of their binding proteins in HSCs and liver tissues by cy/histochemistry; the results showed that GBPs mainly distributed in the cytoplasma membrane and perinuclear region of cytoplasm. The immunocytochemistry was further used to verify some GBPs really exist in Golgi apparatus of the cells. The precision alteration and localization of GBPs referred to pathological changes in HSCs may provide pivotal information to help understand the biological functions of glycans how to exert through their recognition by a wide variety of GBPs. This study could lead to the development of new anti-fibrotic strategies. PMID:26058380

  9. Convergent evolution among immunoglobulin G-binding bacterial proteins.

    PubMed Central

    Frick, I M; Wikström, M; Forsén, S; Drakenberg, T; Gomi, H; Sjöbring, U; Björck, L

    1992-01-01

    Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide, were also inhibited in their interactions with IgGFc by the peptide. Finally, a decapeptide based on a sequence in IgGFc blocked the binding of all three proteins to IgGFc. This unusually clear example of convergent evolution emphasizes the complexity of protein-protein interactions and suggests that bacterial surface-protein interaction with host protein adds selective advantages to the microorganism. Images PMID:1528858

  10. Grating coupled SPR microarray analysis of proteins and cells in blood from mice with breast cancer.

    PubMed

    Mendoza, A; Torrisi, D M; Sell, S; Cady, N C; Lawrence, D A

    2016-01-21

    Biomarker discovery for early disease diagnosis is highly important. Of late, much effort has been made to analyze complex biological fluids in an effort to develop new markers specific for different cancer types. Recent advancements in label-free technologies such as surface plasmon resonance (SPR)-based biosensors have shown promise as a diagnostic tool since there is no need for labeling or separation of cells. Furthermore, SPR can provide rapid, real-time detection of antigens from biological samples since SPR is highly sensitive to changes in surface-associated molecular and cellular interactions. Herein, we report a lab-on-a-chip microarray biosensor that utilizes grating-coupled surface plasmon resonance (GCSPR) and grating-coupled surface plasmon coupled fluorescence (GCSPCF) imaging to detect circulating tumor cells (CTCs) from a mouse model (FVB-MMTV-PyVT). GCSPR and GCSPCF analysis was accomplished by spotting antibodies to surface cell markers, cytokines and stress proteins on a nanofabricated GCSPR microchip and screening blood samples from FVB control mice or FVB-MMTV-PyVT mice with developing mammary carcinomas. A transgenic MMTV-PyVT mouse derived cancer cell line was also analyzed. The analyses indicated that CD24, CD44, CD326, CD133 and CD49b were expressed in both cell lines and in blood from MMTV-PyVT mice. Furthermore, cytokines such as IL-6, IL-10 and TNF-α, along with heat shock proteins HSP60, HSP27, HSc70(HSP73), HSP90 total, HSP70/HSc70, HSP90, HSP70, HSP90 alpha, phosphotyrosine and HSF-1 were overexpressed in MMTV-PyVT mice. PMID:26539568

  11. Fused protein domains inhibit DNA binding by LexA.

    PubMed Central

    Golemis, E A; Brent, R

    1992-01-01

    Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions. Images PMID:1620111

  12. Calmodulin Binding Proteins and Alzheimer’s Disease

    PubMed Central

    O’Day, Danton H.; Eshak, Kristeen; Myre, Michael A.

    2015-01-01

    Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  13. Odorant binding proteins: a biotechnological tool for odour control.

    PubMed

    Silva, Carla; Matamá, Teresa; Azoia, Nuno G; Mansilha, Catarina; Casal, Margarida; Cavaco-Paulo, Artur

    2014-04-01

    The application of an odorant binding protein for odour control and fragrance delayed release from a textile surface was first explored in this work. Pig OBP-1 gene was cloned and expressed in Escherichia coli, and the purified protein was biochemically characterized. The IC₅₀ values (concentrations of competitor that caused a decay of fluorescence to half-maximal intensity) were determined for four distinct fragrances, namely, citronellol, benzyl benzoate, citronellyl valerate and ethyl valerate. The results showed a strong binding of citronellyl valerate, citronellol and benzyl benzoate to the recombinant protein, while ethyl valerate displayed weaker binding. Cationized cotton substrates were coated with porcine odorant binding protein and tested for their capacity to retain citronellol and to mask the smell of cigarette smoke. The immobilized protein delayed the release of citronellol when compared to the untreated cotton. According to a blind evaluation of 30 assessors, the smell of cigarette smoke, trapped onto the fabrics' surface, was successfully attenuated by porcine odorant binding protein (more than 60 % identified the weakest smell intensity after protein exposure compared to β-cyclodextrin-treated and untreated cotton fabrics). This work demonstrated that porcine odorant binding protein can be an efficient solution to prevent and/or remove unpleasant odours trapped on the large surface of textiles. Its intrinsic properties make odorant binding proteins excellent candidates for controlled release systems which constitute a new application for this class of proteins. PMID:24092006

  14. Protein Binding: Do We Ever Learn?▿

    PubMed Central

    Zeitlinger, Markus A.; Derendorf, Hartmut; Mouton, Johan W.; Cars, Otto; Craig, William A.; Andes, David; Theuretzbacher, Ursula

    2011-01-01

    Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein binding in vitro are suggested. PMID:21537013

  15. Therapeutic and analytical applications of arsenic binding to proteins.

    PubMed

    Chen, Beibei; Liu, Qingqing; Popowich, Aleksandra; Shen, Shengwen; Yan, Xiaowen; Zhang, Qi; Li, Xing-Fang; Weinfeld, Michael; Cullen, William R; Le, X Chris

    2015-01-01

    Arsenic binding to proteins plays a pivotal role in the health effects of arsenic. Further knowledge of arsenic binding to proteins will advance the development of bioanalytical techniques and therapeutic drugs. This review summarizes recent work on arsenic-based drugs, imaging of cellular events, capture and purification of arsenic-binding proteins, and biosensing of arsenic. Binding of arsenic to the promyelocytic leukemia fusion oncoprotein (PML-RARα) is a plausible mode of action leading to the successful treatment of acute promyelocytic leukemia (APL). Identification of other oncoproteins critical to other cancers and the development of various arsenicals and targeted delivery systems are promising approaches to the treatment of other types of cancers. Techniques for capture, purification, and identification of arsenic-binding proteins make use of specific binding between trivalent arsenicals and the thiols in proteins. Biarsenical probes, such as FlAsH-EDT2 and ReAsH-EDT2, coupled with tetracysteine tags that are genetically incorporated into the target proteins, are used for site-specific fluorescence labelling and imaging of the target proteins in living cells. These allow protein dynamics and protein-protein interactions to be studied. Arsenic affinity chromatography is useful for purification of thiol-containing proteins, and its combination with mass spectrometry provides a targeted proteomic approach for studying the interactions between arsenicals and proteins in cells. Arsenic biosensors evolved from the knowledge of arsenic resistance and arsenic binding to proteins in bacteria, and have now been developed into analytical techniques that are suitable for the detection of arsenic in the field. Examples in the four areas, arsenic-based drugs, imaging of cellular events, purification of specific proteins, and arsenic biosensors, demonstrate important therapeutic and analytical applications of arsenic protein binding. PMID:25356501

  16. Odorant-Binding Protein: Localization to Nasal Glands and Secretions

    NASA Astrophysics Data System (ADS)

    Pevsner, Jonathan; Sklar, Pamela B.; Snyder, Solomon H.

    1986-07-01

    An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its immunohistochemical localization to mucus-secreting glands of the olfactory and respiratory mucosa. Although OBP was originally isolated as a pyrazine binding protein, both rat and bovine OBP also bind the odorants [3H]methyldihydrojasmonate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3H]methoxypyrazine. We detect substantial odorant-binding activity attributable to OBP in secreted rat nasal mucus and tears but not in saliva, suggesting a role for OBP in transporting or concentrating odorants.

  17. Differentially Expressed RNA from Public Microarray Data Identifies Serum Protein Biomarkers for Cross-Organ Transplant Rejection and Other Conditions

    PubMed Central

    Chen, Rong; Sigdel, Tara K.; Li, Li; Kambham, Neeraja; Dudley, Joel T.; Hsieh, Szu-chuan; Klassen, R. Bryan; Chen, Amery; Caohuu, Tuyen; Morgan, Alexander A.; Valantine, Hannah A.; Khush, Kiran K.; Sarwal, Minnie M.; Butte, Atul J.

    2010-01-01

    Serum proteins are routinely used to diagnose diseases, but are hard to find due to low sensitivity in screening the serum proteome. Public repositories of microarray data, such as the Gene Expression Omnibus (GEO), contain RNA expression profiles for more than 16,000 biological conditions, covering more than 30% of United States mortality. We hypothesized that genes coding for serum- and urine-detectable proteins, and showing differential expression of RNA in disease-damaged tissues would make ideal diagnostic protein biomarkers for those diseases. We showed that predicted protein biomarkers are significantly enriched for known diagnostic protein biomarkers in 22 diseases, with enrichment significantly higher in diseases for which at least three datasets are available. We then used this strategy to search for new biomarkers indicating acute rejection (AR) across different types of transplanted solid organs. We integrated three biopsy-based microarray studies of AR from pediatric renal, adult renal and adult cardiac transplantation and identified 45 genes upregulated in all three. From this set, we chose 10 proteins for serum ELISA assays in 39 renal transplant patients, and discovered three that were significantly higher in AR. Interestingly, all three proteins were also significantly higher during AR in the 63 cardiac transplant recipients studied. Our best marker, serum PECAM1, identified renal AR with 89% sensitivity and 75% specificity, and also showed increased expression in AR by immunohistochemistry in renal, hepatic and cardiac transplant biopsies. Our results demonstrate that integrating gene expression microarray measurements from disease samples and even publicly-available data sets can be a powerful, fast, and cost-effective strategy for the discovery of new diagnostic serum protein biomarkers. PMID:20885780

  18. Actin binding proteins, spermatid transport and spermiation*

    PubMed Central

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  19. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  20. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  1. Selective polyamine-binding proteins. Spermine binding by an androgen-sensitive phosphoprotein.

    PubMed

    Liang, T; Mezzetti, G; Chen, C; Liao, S

    1978-09-01

    Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine greater than thermine greater than greater than putrecine greater than 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein. The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP. The spermine-binding activity of the prostate cytosol protein fraction decreases after castration, but increases very rapidly after the castrated rats are injected with 5alpha-dihydrotestosterone. This finding raises the possibility that, in the postate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin. In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S. PMID:28786

  2. Clinical relevance of drug binding to plasma proteins

    NASA Astrophysics Data System (ADS)

    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  3. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  4. AU-rich RNA binding proteins in hematopoiesis and leukemogenesis.

    PubMed

    Baou, Maria; Norton, John D; Murphy, John J

    2011-11-24

    Posttranscriptional mechanisms are now widely acknowledged to play a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation, and tumorigenesis. Although much attention has focused on microRNAs as regulators of mRNA stability/translation, recent data have highlighted the role of several diverse classes of AU-rich RNA-binding protein in the regulation of mRNA decay/stabilization. AU-rich elements are found in the 3'-untranslated region of many mRNAs that encode regulators of cell growth and survival, such as cytokines and onco/tumor-suppressor proteins. These are targeted by a burgeoning number of different RNA-binding proteins. Three distinct types of AU-rich RNA binding protein (ARE poly-U-binding degradation factor-1/AUF1, Hu antigen/HuR/HuA/ELAVL1, and the tristetraprolin/ZFP36 family of proteins) are essential for normal hematopoiesis. Together with 2 further AU-rich RNA-binding proteins, nucleolin and KHSRP/KSRP, the functions of these proteins are intimately associated with pathways that are dysregulated in various hematopoietic malignancies. Significantly, all of these AU-rich RNA-binding proteins function via an interconnected network that is integrated with microRNA functions. Studies of these diverse types of RNA binding protein are providing novel insight into gene-regulatory mechanisms in hematopoiesis in addition to offering new opportunities for developing mechanism-based targeted therapeutics in leukemia and lymphoma. PMID:21917750

  5. Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

    PubMed

    Xiao, Yongsheng; Wang, Yinsheng

    2016-09-01

    Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:601-619, 2016. PMID:25376990

  6. In Situ Quantification of Protein Binding to the Plasma Membrane

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus. PMID:26039166

  7. The prostate cancer immunome: In silico functional analysis of antigenic proteins from microarray profiling with IgG.

    PubMed

    Luna-Coronell, Johana A; Vierlinger, Klemens; Gamperl, Magdalena; Hofbauer, Johann; Berger, Ingrid; Weinhäusel, Andreas

    2016-04-01

    The study of the immunome of prostate cancer (PCa) and characterization of autoantibody signature from differentially reactive antigens can uncover disease stage proteins, reveal enriched networks and even expose aberrant cellular mechanisms during the disease process. By conducting plasma IgG profiling on protein microarrays presenting 5449 unique human proteins expressed in 15 417 E. coli human cDNA expression clones, we elucidated 471 (21 higher reactive in PCa) differentially reactive antigens in 50 PCa versus 49 patients with benign prostate hyperplasia (BPH) at initial diagnosis. Functional analyzes show that the immune-profile of PCa compared to BPH control samples is significantly enriched in features targeting Cellular assembly, Cell death and pathways involved in Cell cycle, translation, and assembly of proteins as EIF2 signaling, PCa related genes as AXIN1 and TP53, and ribosomal proteins (e.g. RPS10). An overlap of 61 (out of 471) DIRAGs with the published 1545 antigens from the SEREX database has been found, however those were higher reactive in BPH. Clinical relevance is shown when antibody-reactivities against eight proteins were significantly (p < 0.001) correlated with Gleason-score. Herewith we provide a biological and pathophysiological characterization of the immunological layer of cancerous (PCa) versus benign (BPH) disease, derived from antibody profiling on protein microarrays. PMID:27089054

  8. A protocol for the systematic and quantitative measurement of protein-lipid interactions using the liposome-microarray-based assay.

    PubMed

    Saliba, Antoine-Emmanuel; Vonkova, Ivana; Deghou, Samy; Ceschia, Stefano; Tischer, Christian; Kugler, Karl G; Bork, Peer; Ellenberg, Jan; Gavin, Anne-Claude

    2016-06-01

    Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales. PMID:27149326

  9. Protein-protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes.

    PubMed

    Hanes, Melinda S; Ratcliff, Kathleen; Marqusee, Susan; Handel, Tracy M

    2010-10-01

    Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein-ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein-protein complex involving the β-lactamase TEM-1 and various β-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C(m), of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein-protein complexes. From a small set (n = 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC(m) was observed. From this "calibration curve," accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived ΔC(m) values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies. PMID:20669180

  10. Odorant-binding proteins from a primitive termite.

    PubMed

    Ishida, Yuko; Chiang, Vicky P; Haverty, Michael I; Leal, Walter S

    2002-09-01

    Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths. PMID:12449514

  11. Search for Amyloid-Binding Proteins by Affinity Chromatography

    PubMed Central

    Calero, Miguel; Rostagno, Agueda; Ghiso, Jorge

    2013-01-01

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

  12. A Comparison between Manual and Automated Evaluations of Tissue Microarray Patterns of Protein Expression

    PubMed Central

    Alvarenga, Arthur W.; Coutinho-Camillo, Claudia M.; Rodrigues, Bruna R.; Rocha, Rafael M.; Torres, Luiz Fernando B.; Martins, Vilma R.; da Cunha, Isabela W.

    2013-01-01

    Tissue microarray technology enables us to evaluate the pattern of protein expression in large numbers of samples. However, manual data acquisition and analysis still represent a challenge because they are subjective and time-consuming. Automated analysis may thus increase the speed and reproducibility of evaluation. However, the reliability of automated analysis systems should be independently evaluated. Herein, the expression of phosphorylated AKT and mTOR was determined by ScanScope XT (Aperio; Vista, CA) and ACIS III (Dako; Glostrup, Denmark) and compared with the manual analysis by two observers. The percentage of labeled pixels or nuclei analysis had a good correlation between human observers and automated systems (κ = 0.855 and 0.879 for ScanScope vs. observers and κ = 0.765 and 0.793 for ACIS III vs. observers). The intensity of labeling determined by ScanScope was also correlated with that found by the human observers (correlation index of 0.946 and 0.851 for pAKT and 0.851 and 0.875 for pmTOR). However, the correlation between ACIS III and human observation varied for labeling intensity and was considered poor in some cases (correlation index of 0.718 and 0.680 for pAKT and 0.223 and 0.225 for pmTOR). Thus, the percentage of positive pixels or nuclei determination was satisfactorily performed by both systems; however, labeling intensity was better identified by ScanScope XT. PMID:23340270

  13. The actin binding protein adseverin regulates osteoclastogenesis.

    PubMed

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  14. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    PubMed Central

    Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  15. The RNA binding protein RBM38 (RNPC1) regulates splicing during late erythroid differentiation.

    PubMed

    Heinicke, Laurie A; Nabet, Behnam; Shen, Shihao; Jiang, Peng; van Zalen, Sebastiaan; Cieply, Benjamin; Russell, J Eric; Xing, Yi; Carstens, Russ P

    2013-01-01

    Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71(+) erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation. PMID:24250749

  16. The RNA Binding Protein RBM38 (RNPC1) Regulates Splicing during Late Erythroid Differentiation

    PubMed Central

    Heinicke, Laurie A.; Nabet, Behnam; Shen, Shihao; Jiang, Peng; van Zalen, Sebastiaan; Cieply, Benjamin; Russell, J. Eric; Xing, Yi; Carstens, Russ P.

    2013-01-01

    Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71+ erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation. PMID:24250749

  17. Concentration-dependent Cu(II) binding to prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

  18. Cooperative binding modes of Cu(II) in prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  19. Binding capability of the enediyne-associated apoprotein to human tumors and constitution of a ligand oligopeptide-integrated protein.

    PubMed

    Cai, Lin; Chen, Hongxia; Miao, Qingfang; Wu, Shuying; Shang, Yue; Zhen, Yongsu

    2009-10-26

    The molecule of lidamycin that belongs to the chromoprotein family of antitumor antibiotics is composed of an apoprotein (LDP) and an enediyne chromophore. The enediyne moiety of the molecule is responsible for the potent cytotoxicity; however, the biological function of the apoprotein moiety, particularly its interaction with cancer cells, remains unclear. In present study, the binding capability of LDP to human tumors was detected for the first time by tissue microarray. LDP bound to various human tumors with significant difference from the corresponding normal tissues. Positive correlation between binding activity and the overexpression of VEGF and EGFR was confirmed by lung carcinoma tissue microarray. A fusion protein LG-LDP that consists of LDP and a ligand oligopeptide to EGFR was constructed by DNA recombination. LG-LDP showed augmented binding to EGFR-overexpressing cancer cells. Furthermore, an energized fusion protein LG-LDP-AE was prepared by integrating the active enediyne (AE) into LG-LDP molecule. By MTT assay, LG-LDP-AE displayed extremely potent cytotoxicity to cancer cells with IC50 approximate to 0.01nM. The results indicate that LDP binds to various human tumors and it might serve as a delivery carrier by integration of ligand oligopeptide to manufacture motif-based, targeted fusion proteins for cancer. PMID:19737585

  20. Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

    PubMed Central

    Samuels, M; Deshpande, G; Schedl, P

    1998-01-01

    The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain. PMID:9592147

  1. A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays

    PubMed Central

    2012-01-01

    Background DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin. Results For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform. Conclusions To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  3. EMSA Analysis of DNA Binding By Rgg Proteins

    PubMed Central

    LaSarre, Breah; Federle, Michael J.

    2016-01-01

    In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases).

  4. Guardian of Genetic Messenger-RNA-Binding Proteins

    PubMed Central

    Anji, Antje; Kumari, Meena

    2016-01-01

    RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins. PMID:26751491

  5. Purification of a Zn-binding phloem protein with sequence identity to chitin-binding proteins.

    PubMed Central

    Taylor, K C; Albrigo, L G; Chase, C D

    1996-01-01

    In citrus blight, a decline disorder of unknown etiology, the tree canopy exhibits symptoms of Zn deficiency while Zn accumulates in the trunk phloem. We have purified a Zn-binding protein (ZBP) from phloem tissue of healthy and blight-affected citrus (Citrus sinensis [L.] Osbeck on Citrus jambhiri [L.]). The molecular weight of the ZBP was estimated to be 5000 by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion-exchange chromatography at pH 8.0 demonstrated the 5-kD ZBP to be anionic. A partial N-terminal amino acid sequence revealed a cysteine-, glycine-rich domain with 45 to 80% identity with the chitin-binding domain of hevein, wheat germ agglutinin, and several class I chitinases. That the abundance of this protein increased 2.5-fold in association with Zn accumulation in the phloem is characteristic of citrus blight. Tissue mass changes of the phloem suggests that altered tissue structure accompanies blight. Phloem accumulation of the 5-kD ZBP may be in response to wounding or other stress of blight-affected citrus. PMID:8742339

  6. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    PubMed

    Khan, Waqasuddin; Duffy, Fergal; Pollastri, Gianluca; Shields, Denis C; Mooney, Catherine

    2013-01-01

    Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif) containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58).Next, we trained a bidirectional recurrent neural network (BRNN) using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72) showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods) clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors. PMID:24019881

  7. Identification of AOSC-binding proteins in neurons

    NASA Astrophysics Data System (ADS)

    Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

    2008-11-01

    Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

  8. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. PMID:26873273

  9. The human "magnesome": detecting magnesium binding sites on human proteins

    PubMed Central

    2012-01-01

    Background Magnesium research is increasing in molecular medicine due to the relevance of this ion in several important biological processes and associated molecular pathogeneses. It is still difficult to predict from the protein covalent structure whether a human chain is or not involved in magnesium binding. This is mainly due to little information on the structural characteristics of magnesium binding sites in proteins and protein complexes. Magnesium binding features, differently from those of other divalent cations such as calcium and zinc, are elusive. Here we address a question that is relevant in protein annotation: how many human proteins can bind Mg2+? Our analysis is performed taking advantage of the recently implemented Bologna Annotation Resource (BAR-PLUS), a non hierarchical clustering method that relies on the pair wise sequence comparison of about 14 millions proteins from over 300.000 species and their grouping into clusters where annotation can safely be inherited after statistical validation. Results After cluster assignment of the latest version of the human proteome, the total number of human proteins for which we can assign putative Mg binding sites is 3,751. Among these proteins, 2,688 inherit annotation directly from human templates and 1,063 inherit annotation from templates of other organisms. Protein structures are highly conserved inside a given cluster. Transfer of structural properties is possible after alignment of a given sequence with the protein structures that characterise a given cluster as obtained with a Hidden Markov Model (HMM) based procedure. Interestingly a set of 370 human sequences inherit Mg2+ binding sites from templates sharing less than 30% sequence identity with the template. Conclusion We describe and deliver the "human magnesome", a set of proteins of the human proteome that inherit putative binding of magnesium ions. With our BAR-hMG, 251 clusters including 1,341 magnesium binding protein structures

  10. Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

    PubMed Central

    Shi, Yanbo; Harvey, Ian; Campopiano, Dominic; Sadler, Peter J.

    2010-01-01

    Ferric ion binding proteins (Fbps) transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III) is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues) together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed. PMID:20445753

  11. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  12. The Potassium Binding Protein Kbp Is a Cytoplasmic Potassium Sensor.

    PubMed

    Ashraf, Khuram U; Josts, Inokentijs; Mosbahi, Khedidja; Kelly, Sharon M; Byron, Olwyn; Smith, Brian O; Walker, Daniel

    2016-05-01

    Escherichia coli possesses a number of specific K(+) influx and efflux systems that maintain an appropriate intracellular K(+) concentration. Although regulatory mechanisms have been identified for a number of these transport systems, the exact mechanism through which K(+) concentration is sensed in the cell remains unknown. In this work we show that Kbp (K(+) binding protein, formerly YgaU), a soluble 16-kDa cytoplasmic protein from Escherichia coli, is a highly specific K(+) binding protein and is required for normal growth in the presence of high levels of external K(+). Kbp binds a single potassium ion with high specificity over Na(+) and other metal ions found in biological systems, although, in common with K(+) transporters, it also binds Rb(+) and Cs(+). Dissection of the K(+) binding determinants of Kbp suggests a mechanism through which Kbp is able to sense changes in K(+) concentration over the relevant range of intracellular K(+) concentrations. PMID:27112601

  13. Serial Analysis of 38 Proteins during the Progression of Human Breast Tumor in Mice Using an Antibody Colocalization Microarray*

    PubMed Central

    Li, Huiyan; Bergeron, Sébastien; Annis, Matthew G.; Siegel, Peter M.; Juncker, David

    2015-01-01

    Proteins in serum or plasma hold great potential for use in disease diagnosis and monitoring. However, the correlation between tumor burden and protein biomarker concentration has not been established. Here, using an antibody colocalization microarray, the protein concentration in serum was measured and compared with the size of mammary xenograft tumors in 11 individual mice from the time of injection; seven blood samples were collected from each tumor-bearing mouse as well as control mice on a weekly basis. The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor. To evaluate the diagnosis of cancer based on these proteins using either an absolute threshold (i.e. a concentration cutoff) or self-referenced differential threshold based on the increase in concentration before cell injection, receiver operating characteristic curves were produced for 10 proteins with increased concentration, and the area under curve was calculated for each time point based on a single protein or on a panel of proteins, in each case showing a rapid increase of the area under curve. Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2. These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models. PMID:25680959

  14. Serial analysis of 38 proteins during the progression of human breast tumor in mice using an antibody colocalization microarray.

    PubMed

    Li, Huiyan; Bergeron, Sébastien; Annis, Matthew G; Siegel, Peter M; Juncker, David

    2015-04-01

    Proteins in serum or plasma hold great potential for use in disease diagnosis and monitoring. However, the correlation between tumor burden and protein biomarker concentration has not been established. Here, using an antibody colocalization microarray, the protein concentration in serum was measured and compared with the size of mammary xenograft tumors in 11 individual mice from the time of injection; seven blood samples were collected from each tumor-bearing mouse as well as control mice on a weekly basis. The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor. To evaluate the diagnosis of cancer based on these proteins using either an absolute threshold (i.e. a concentration cutoff) or self-referenced differential threshold based on the increase in concentration before cell injection, receiver operating characteristic curves were produced for 10 proteins with increased concentration, and the area under curve was calculated for each time point based on a single protein or on a panel of proteins, in each case showing a rapid increase of the area under curve. Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2. These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models. PMID:25680959

  15. The RNA binding site of bacteriophage MS2 coat protein.

    PubMed Central

    Peabody, D S

    1993-01-01

    The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat. Images PMID:8440248

  16. Exchange Kinetics of a Hydrophobic Ligand Binding Protein

    NASA Astrophysics Data System (ADS)

    Vaughn, Jeff; Stone, Martin

    2002-03-01

    Conformational fluctuations of proteins are thought to be important for determining the functional roles in biological activity. In some cases, the rates of these conformational changes may be directly correlated to, for example, the rates of catalysis or ligand binding. We are studying the role of conformational fluctuations in the binding of small volatile hydrophobic pheromones by the mouse major urinary proteins (MUPs). Communication among mice occurs, in part, with the MUP-1 protein. This urinary protein binds pheromones as a way to increase the longevity of the pheromone in an extracellular environment. Of interest is that the crystal structure of MUP-1 with a pheromone ligand shows the ligand to be completely occluded from the solvent with no obvious pathway to enter or exit. This suggests that conformational exchange of the protein may be required for ligand binding and release to occur. We hypothesize that the rate of conformational exchange may be a limiting factor determining the rate of ligand association and dissociation. By careful measurement of the on- and off-rates of ligand binding and the rates of conformational changes of the protein, a more defined picture of the interplay between protein structure and function can be obtained. To this end, heteronuclear saturation transfer, ^15N-exchange and ^15N dynamics experiments have been employed to probe the kinetics of ligand binding to MUP-1.

  17. General RNA binding proteins render translation cap dependent.

    PubMed Central

    Svitkin, Y V; Ovchinnikov, L P; Dreyfuss, G; Sonenberg, N

    1996-01-01

    Translation in rabbit reticulocyte lysate is relatively independent of the presence of the mRNA m7G cap structure and the cap binding protein, eIF-4E. In addition, initiation occurs frequently at spurious internal sites. Here we show that a critical parameter which contributes to cap-dependent translation is the amount of general RNA binding proteins in the extract. Addition of several general RNA binding proteins, such as hnRNP A1, La autoantigen, pyrimidine tract binding protein (hnRNP I/PTB) and the major core protein of cytoplasmic mRNP (p50), rendered translation in a rabbit reticulocyte lysate cap dependent. These proteins drastically inhibited the translation of an uncapped mRNA, but had no effect on translation of a capped mRNA. Based on these and other results, we suggest that one function of general mRNA binding proteins in the cytoplasm is to promote ribosome binding by a 5' end, cap-mediated mechanism, and prevent spurious initiations at aberrant translation start sites. Images PMID:9003790

  18. Identification of multiple salicylic acid-binding proteins using two high throughput screens

    PubMed Central

    Manohar, Murli; Tian, Miaoying; Moreau, Magali; Park, Sang-Wook; Choi, Hyong Woo; Fei, Zhangjun; Friso, Giulia; Asif, Muhammed; Manosalva, Patricia; von Dahl, Caroline C.; Shi, Kai; Ma, Shisong; Dinesh-Kumar, Savithramma P.; O'Doherty, Inish; Schroeder, Frank C.; van Wijk, Klass J.; Klessig, Daniel F.

    2014-01-01

    Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays. PMID:25628632

  19. Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms.

    PubMed

    Chou, Shan-Ho; Galperin, Michael Y

    2016-01-01

    Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms. PMID:26055114

  20. Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

    PubMed Central

    2015-01-01

    ABSTRACT Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms. PMID:26055114

  1. Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro.

    PubMed

    Pasternack, M S; Bleier, K J; McInerney, T N

    1991-08-01

    The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells. PMID:1860869

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

    PubMed

    Hunter, S A; Cochran, J R

    2016-01-01

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

  3. Ice-shell purification of ice-binding proteins.

    PubMed

    Marshall, Craig J; Basu, Koli; Davies, Peter L

    2016-06-01

    Ice-affinity purification is a simple and efficient method of purifying to homogeneity both natural and recombinant ice-binding proteins. The purification involves the incorporation of ice-binding proteins into slowly-growing ice and the exclusion of other proteins and solutes. In previous approaches, the ice was grown around a hollow brass finger through which coolant was circulated. We describe here an easily-constructed apparatus that employs ice affinity purification that not only shortens the time for purification from 1-2 days to 1-2 h, but also enhances yield and purity. In this apparatus, the surface area for the separation was increased by extracting the ice-binding proteins into an ice-shell formed inside a rotating round-bottom flask partially submerged in a sub-zero bath. In principle, any ice-binding compound can be recovered from liquid solution, and the method is readily scalable. PMID:27025155

  4. Erythrocyte Protein 4.1 Binds and Regulates Myosin

    NASA Astrophysics Data System (ADS)

    Pasternack, Gary R.; Racusen, Richard H.

    1989-12-01

    Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

  5. Theoretical studies of protein-protein and protein-DNA binding rates

    NASA Astrophysics Data System (ADS)

    Alsallaq, Ramzi A.

    Proteins are folded chains of amino acids. Some of the amino acids (e.g. Lys, Arg, His, Asp, and Glu) carry charges under physiological conditions. Proteins almost always function through binding to other proteins or ligands, for example barnase is a ribonuclease protein, found in the bacterium Bacillus amyloliquefaceus. Barnase degrades RNA by hydrolysis. For the bacterium to inhibit the potentially lethal action of Barnase within its own cell it co-produces another protein called barstar which binds quickly, and tightly, to barnase. The biological function of this binding is to block the active site of barnase. The speeds (rates) at which proteins associate are vital to many biological processes. They span a wide range (from less than 103 to 108 M-1s-1 ). Rates greater than ˜ 106 M -1s-1 are typically found to be manifestations of enhancements by long-range electrostatic interactions between the associating proteins. A different paradigm appears in the case of protein binding to DNA. The rate in this case is enhanced through attractive surface potential that effectively reduces the dimensionality of the available search space for the diffusing protein. This thesis presents computational and theoretical models on the rate of association of ligands/proteins to other proteins or DNA. For protein-protein association we present a general strategy for computing protein-protein rates of association. The main achievements of this strategy is the ability to obtain a stringent reaction criteria based on the landscape of short-range interactions between the associating proteins, and the ability to compute the effect of the electrostatic interactions on the rates of association accurately using the best known solvers for Poisson-Boltzmann equation presently available. For protein-DNA association we present a mathematical model for proteins targeting specific sites on a circular DNA topology. The main achievements are the realization that a linear DNA with reflecting ends

  6. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    PubMed

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  7. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  8. Therapeutic potential of vitamin D-binding protein.

    PubMed

    Gomme, Peter T; Bertolini, Joseph

    2004-07-01

    Vitamin D-binding protein (DBP) is a multi-functional plasma protein with many important functions. These include transport of vitamin D metabolites, control of bone development, binding of fatty acids, sequestration of actin and a range of less-defined roles in modulating immune and inflammatory responses. Exploitation of the unique properties of DBP could enable the development of important therapeutic agents for the treatment of a variety of diseases. PMID:15245906

  9. Plasma protein binding of nitroxynil in several species.

    PubMed

    Alvinerie, M; Floc'h, R; Galtier, P

    1991-06-01

    The binding of nitroxynil to total plasma proteins of cows, sheep and rabbits was characterized using equilibrium dialysis. The data indicate clearly that nitroxynil was highly (97-98%) bound to plasma protein of each animal. This linear binding would be due to the particular power exerted by serum albumin. The results are in good agreement with known pharmacokinetic properties of nitroxynil in domestic species. PMID:1920604

  10. Subcellular distribution of small GTP binding proteins in pancreas: Identification of small GTP binding proteins in the rough endoplasmic reticulum

    SciTech Connect

    Nigam, S.K. )

    1990-02-01

    Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5{prime}-({gamma}-({sup 35}S)thio)triphosphate (GTP({gamma}-{sup 35}S)) was assayed in each fraction. Enrichment of GTP({gamma}-{sup 35}S) binding was greatest in the interfacial smooth microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a ({alpha}-{sup 32}P)GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP({gamma}-{sup 35}S) binding, as well as the small GTP binding proteins detected by the ({alpha}-{sup 32}P)GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMS were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP({gamma}-{sup 35}S) binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.

  11. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    PubMed

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  12. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  13. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  14. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  15. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  16. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  17. Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

    PubMed Central

    Tran, Giao V. Q.; Luong, Trang T. D.; Park, Eun-Mee; Kim, Jong-Wook; Choi, Jae-Woong; Park, Chorong; Lim, Yun-Sook

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for virus propagation. To identify the cellular factors involved in HCV propagation, we recently performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of 90 cellular protein candidates, we selected the soluble resistance-related calcium-binding protein (sorcin) for further characterization. Sorcin is a calcium-binding protein and is highly expressed in certain cancer cells. We verified that NS5A interacted with sorcin through domain I of NS5A, and phosphorylation of the threonine residue 155 of sorcin played a crucial role in protein interaction. Small interfering RNA (siRNA)-mediated knockdown of sorcin impaired HCV propagation. Silencing of sorcin expression resulted in a decrease of HCV assembly without affecting HCV RNA and protein levels. We further demonstrated that polo-like kinase 1 (PLK1)-mediated phosphorylation of sorcin was increased by NS5A. We showed that both phosphorylation and calcium-binding activity of sorcin were required for HCV propagation. These data indicate that HCV modulates sorcin activity via NS5A protein for its own propagation. IMPORTANCE Sorcin is a calcium-binding protein and regulates intracellular calcium homeostasis. HCV NS5A interacts with sorcin, and phosphorylation of sorcin is required for protein interaction. Gene silencing of sorcin impaired HCV propagation at the assembly step of the HCV life cycle. Sorcin is phosphorylated by PLK1 via protein interaction. We showed that sorcin interacted with both NS5A and PLK1, and PLK1-mediated phosphorylation of sorcin was increased by NS5A. Moreover, calcium-binding activity of sorcin played a crucial role in HCV propagation. These data provide evidence that HCV regulates host calcium metabolism for virus propagation, and thus manipulation of sorcin activity may represent a novel therapeutic target for HCV. PMID:26719254

  18. Being a binding site: characterizing residue composition of binding sites on proteins.

    PubMed

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-01-01

    The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs. PMID:18305831

  19. Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

    PubMed Central

    Wen, Xuexia; Bao, Hongmei; Shi, Lin; Tao, Qimeng; Jiang, Yongping; Zeng, Xianying; Xu, Xiaolong; Tian, Guobin; Zheng, Shimin; Chen, Hualan

    2016-01-01

    Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza. PMID:26938453

  20. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    PubMed

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted. PMID:26028028

  1. Protein-DNA binding in high-resolution

    PubMed Central

    Mahony, Shaun; Pugh, B. Franklin

    2015-01-01

    Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATACseq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases, and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches. PMID:26038153

  2. Discovery and validation of an INflammatory PROtein-driven GAstric cancer Signature (INPROGAS) using antibody microarray-based oncoproteomics

    PubMed Central

    Puig-Costa, Manuel; Codina-Cazador, Antonio; Cortés-Pastoret, Elisabet; Oliveras-Ferraros, Cristina; Cufí, Sílvia; Flaquer, Sílvia; Llopis-Puigmarti, Francesca; Pujol-Amado, Eulalia; Corominas-Faja, Bruna; Cuyàs, Elisabet; Ortiz, Rosa; Lopez-Bonet, Eugeni; Queralt, Bernardo; Guardeño, Raquel; Martin-Castillo, Begoña; Roig, Josep; Joven, Jorge; Menendez, Javier A.

    2014-01-01

    This study aimed to improve gastric cancer (GC) diagnosis by identifying and validating an INflammatory PROtein-driven GAstric cancer Signature (hereafter INPROGAS) using low-cost affinity proteomics. The detection of 120 cytokines, 43 angiogenic factors, 41 growth factors, 40 inflammatory factors and 10 metalloproteinases was performed using commercially available human antibody microarray-based arrays. We identified 21 inflammation-related proteins (INPROGAS) with significant differences in expression between GC tissues and normal gastric mucosa in a discovery cohort of matched pairs (n=10) of tumor/normal gastric tissues. Ingenuity pathway analysis confirmed the “inflammatory response”, “cellular movement” and “immune cell trafficking” as the most overrepresented biofunctions within INPROGAS. Using an expanded independent validation cohort (n = 22), INPROGAS classified gastric samples as “GC” or “non-GC” with a sensitivity of 82% (95% CI 59-94) and a specificity of 73% (95% CI 49-89). The positive predictive value and negative predictive value in this validation cohort were 75% (95% CI 53-90) and 80% (95% CI 56-94), respectively. The positive predictive value and negative predictive value in this validation cohort were 75% (95% CI 53-90) and 80% (95% CI 56-94), respectively. Antibody microarray analyses of the GC-associated inflammatory proteome identified a 21-protein INPROGAS that accurately discriminated GC from noncancerous gastric mucosa. PMID:24722433

  3. Binding of CCAAT displacement protein CDP to adenovirus packaging sequences.

    PubMed

    Erturk, Ece; Ostapchuk, Philomena; Wells, Susanne I; Yang, Jihong; Gregg, Keqin; Nepveu, Alain; Dudley, Jaquelin P; Hearing, Patrick

    2003-06-01

    Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process. PMID:12743282

  4. Computational evaluation of protein – small molecule binding

    PubMed Central

    Guvench, Olgun; MacKerell, Alexander D.

    2009-01-01

    Determining protein – small molecule binding affinity is a key component of present-day rational drug discovery. To circumvent the time, labor, and materials costs associated with experimental protein – small molecule binding assays, a variety of structure-based computational methods have been developed for determining protein – small molecule binding affinities. These methods can be placed in one of two classes: accurate but slow (Class 1), and fast but approximate (Class 2). Class 1 methods, which explicitly take into account protein flexibility and include an atomic-level description of solvation, are capable of quantitatively reproducing experimental protein – small molecule absolute binding free energies. However, Class 1 computational requirements make screening thousands to millions of small molecules against a protein, as required for rational drug design, infeasible for the foreseeable future. Class 2 methods, on the other hand, are sufficiently fast to perform such inhibitor screening, yet they suffer from limited descriptions of protein flexibility and solvation, which in turn limit their ability to select and rank-order small molecules by computed binding affinities. This review presents an overview of Class 1 and Class 2 methods, avenues of research in Class 2 methods aimed at bringing them closer to Class 1 accuracy, and intermediate approaches that incorporate features of both Class 1 and Class 2 methods. PMID:19162472

  5. Lipid A binding proteins in macrophages detected by ligand blotting

    SciTech Connect

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

    1987-05-01

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.

  6. Metal-binding proteins as metal pollution indicators

    SciTech Connect

    Hennig, H.F.

    1986-03-01

    The fact that metal-binding proteins are a consequence of elevated metal concentration in organisms is well known. What has been overlooked is that the presence of these proteins provides a unique opportunity to reformulate the criteria of metal pollution. The detoxification effect of metal-binding proteins in animals from polluted areas has been cited, but there have been only very few studies relating metal-binding proteins to pollution. This lack is due partly to the design of most experiments, which were aimed at isolation of metal-binding proteins and hence were of too short duration to allow for correlation to adverse physiological effects on the organism. In this study metal-binding proteins were isolated and characterized from five different marine animals (rock lobster, Jasus lalandii; hermit crab, Diogenes brevirostris; sandshrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis; and limpet, Patella granularis). These animals were kept under identical metal-enriched conditions, hence eliminating differences in method and seasons. The study animals belonged to different phyla; varied in size, mass, age, behavior, food requirements and life stages; and accumulated metals at different rates. It is possible to link unseasonal moulting in crustacea, a known physiological effect due to a metal-enriched environment, to the production of the metal-binding protein without evidence of obvious metal body burden. Thus a new concept of pollution is defined: the presence of metal-binding proteins confirms toxic metal pollution. This concept was then tested under field conditions in the whelk Bullia digitalis and in metal-enriched grass.

  7. A Correlation between Protein Function and Ligand Binding Profiles

    PubMed Central

    Shortridge, Matthew D.; Bokemper, Michael; Copeland, Jennifer C.; Stark, Jaime L.; Powers, Robert

    2011-01-01

    We report that proteins with the same function bind the same set of small molecules from a standardized chemical library. This observation led to a quantifiable and rapidly adaptable method for protein functional analysis using experimentally-derived ligand binding profiles. Ligand binding is measured using a high-throughput NMR ligand affinity screen with a structurally diverse chemical library. The method was demonstrated using a set of 19 proteins with a range of functions. A statistically significant similarity in ligand binding profiles was only observed between the two functionally identical albumins and between the five functionally similar amylases. This new approach is independent of sequence, structure or evolutionary information, and therefore, extends our ability to analyze and functionally annotate novel genes. PMID:21366353

  8. Binding profile of spiramycin to oviducal proteins of laying hens.

    PubMed

    Furusawa, N

    2000-12-01

    In vitro protein binding of spiramycin (SP) in the plasma and oviducts of laying hens was studied. The data for SP were compared with those for oxytetracycline (OTC), sulphadimidine (SDD), sulphamonomethoxine (SMM) and sulphaquinoxaline (SQ). The two oviduct segments, magnum (M) and isthmus plus shell gland (IS), were collected. The soluble (cell sap) fractions from the magnum (M-S9) and the isthmus plus shell gland (IS-S9) were used as samples. Plasma protein binding was highest for SQ (81.4%) (P < 0.01), and lowest for SDD (30.9%) (P < 0.01). No M-S9 protein binding of OTC was found. The IS-S9 protein binding of SP (60.4%) was very much higher than those of OTC (0.8%), SDD (4.1%), SMM (4.0%) and SQ (12.3%) (P < 0.01). Biological half-lives of these drugs in egg albumen were directly correlated to the extent of their binding to IS proteins. Of plasma, M-S9 and IS-S9, variation in SP concentration in the ranges from 1 to 20 micrograms/ml did not alter the binding properties of the drug. PMID:11199206

  9. Detecting O2 binding sites in protein cavities

    PubMed Central

    Kitahara, Ryo; Yoshimura, Yuichi; Xue, Mengjun; Kameda, Tomoshi; Mulder, Frans A. A.

    2016-01-01

    Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in 1H, 15N, and 13C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins. PMID:26830762

  10. Assessing Energetic Contributions to Binding from a Disordered Region in a Protein-Protein Interaction

    SciTech Connect

    S Cho; C Swaminathan; D Bonsor; M Kerzic; R Guan; J Yang; C Kieke; P Anderson; D Kranz; et al.

    2011-12-31

    Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

  11. Preoperative overnight parenteral nutrition (TPN) improves skeletal muscle protein metabolism indicated by microarray algorithm analyses in a randomized trial.

    PubMed

    Iresjö, Britt-Marie; Engström, Cecilia; Lundholm, Kent

    2016-06-01

    Loss of muscle mass is associated with increased risk of morbidity and mortality in hospitalized patients. Uncertainties of treatment efficiency by short-term artificial nutrition remain, specifically improvement of protein balance in skeletal muscles. In this study, algorithmic microarray analysis was applied to map cellular changes related to muscle protein metabolism in human skeletal muscle tissue during provision of overnight preoperative total parenteral nutrition (TPN). Twenty-two patients (11/group) scheduled for upper GI surgery due to malignant or benign disease received a continuous peripheral all-in-one TPN infusion (30 kcal/kg/day, 0.16 gN/kg/day) or saline infusion for 12 h prior operation. Biopsies from the rectus abdominis muscle were taken at the start of operation for isolation of muscle RNA RNA expression microarray analyses were performed with Agilent Sureprint G3, 8 × 60K arrays using one-color labeling. 447 mRNAs were differently expressed between study and control patients (P < 0.1). mRNAs related to ribosomal biogenesis, mRNA processing, and translation were upregulated during overnight nutrition; particularly anabolic signaling S6K1 (P < 0.01-0.1). Transcripts of genes associated with lysosomal degradation showed consistently lower expression during TPN while mRNAs for ubiquitin-mediated degradation of proteins as well as transcripts related to intracellular signaling pathways, PI3 kinase/MAPkinase, were either increased or decreased. In conclusion, muscle mRNA alterations during overnight standard TPN infusions at constant rate altered mRNAs associated with mTOR signaling; increased initiation of protein translation; and suppressed autophagy/lysosomal degradation of proteins. This indicates that overnight preoperative parenteral nutrition is effective to promote muscle protein metabolism. PMID:27273879

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

    PubMed

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

    2015-01-01

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

  13. Mapping the Ligand-Binding Region of Borrelia hermsii Fibronectin-Binding Protein

    PubMed Central

    Brenner, Christiane; Bomans, Katharina; Habicht, Jüri; Simon, Markus M.; Wallich, Reinhard

    2013-01-01

    Many pathogenic microorganisms express fibronectin-binding molecules that facilitate their adherence to the extracellular matrix and/or entry into mammalian cells. We have previously described a Borrelia recurrentis gene, cihC that encodes a 40-kDa surface receptor for both, fibronectin and the complement inhibitors C4bp and C1-Inh. We now provide evidence for the expression of a group of highly homologues surface proteins, termed FbpA, in three B. hermsii isolates and two tick-borne relapsing fever spirochetes, B. parkeri and B. turicatae. When expressed in Escherichia coli or B. burgdorferi, four out of five proteins were shown to selectively bind fibronectin, whereas none of five proteins were able to bind the human complement regulators, C4bp and C1-Inh. By applying deletion mutants of the B. hermsii fibronectin-binding proteins a putative high-affinity binding site for fibronectin was mapped to its central region. In addition, the fibronectin-binding proteins of B. hermsii were found to share sequence homology with BBK32 of the Lyme disease spirochete B. burgdorferi with similar function suggesting its involvement in persistence and/or virulence of relapsing fever spirochetes. PMID:23658828

  14. Perturbation Approaches for Exploring Protein Binding Site Flexibility to Predict Transient Binding Pockets.

    PubMed

    Kokh, Daria B; Czodrowski, Paul; Rippmann, Friedrich; Wade, Rebecca C

    2016-08-01

    Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design. PMID:27399277

  15. Detergent binding as a sensor of hydrophobicity and polar interactions in the binding cavities of proteins.

    PubMed

    Peyre, Véronique; Lair, Virginie; André, Virginie; le Maire, Guerric; Kragh-Hansen, Ulrich; le Maire, Marc; Møller, Jesper V

    2005-09-13

    To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein-ligand binding, we studied the interaction of human serum albumin (HSA) and beta-lactoglobulin with various aliphatic (C10-C14) cationic and zwitterionic detergents. We find that cationic detergents, at levels that do not cause unfolding, interact with a single site on beta-lactoglobulin and with two primary and five to six secondary sites on HSA with an affinity that is approximately the same as that with which zwitterionic (dimethylamineoxide) detergents interact, suggesting the absence of significant electrostatic interactions in the high-affinity binding of these compounds. The binding affinity for all of the groups of compounds was dependent upon hydrocarbon chain length, suggesting the predominant role of hydrophobic forces, supported by polar interactions at the protein surface. A distinct correlation between the binding energy and the propensity for micelle formation within the group of cationic or noncharged (nonionic and zwitterionic) detergents indicated that the critical micellar concentration (CMC) for each of these detergent groups, rather than the absolute length of the hydrocarbon chain, can be used to compare their hydrophobicities during their interaction with protein. Intrinsic fluorescence data suggest that the two primary binding sites on serum albumin for the zwitterionic and cationic compounds are located in the C-terminal part of the albumin molecule, possibly in the Sudlow II binding region. Comparisons with previous binding data on anionic amphiphiles emphasize the important contribution of ion bond formation and other polar interactions in the binding of fatty acids and dodecyl sulfate (SDS) by HSA but not by beta-lactoglobulin. Electrostatic interactions by cationic detergents played a significant role in destabilizing the protein structure at high binding levels, with beta-lactoglobulin being more susceptible to unfolding than HSA. Zwitterionic

  16. Studies on the spermatogenic sulfogalactolipid binding protein SLIP 1

    SciTech Connect

    Lingwood, C.; Nutikka, A. )

    1991-02-01

    We have purified the testicular sulfogalactolipid binding protein SLIP 1 and shown by photoaffinity labeling that it contains an ATP binding site. Purified SLIP 1 was fluorescently labeled and shown to retain specific sulfogalactolipid binding function. This probe was used to investigate the topology of SLIP 1 binding sites on testicular germ cells. The binding pattern precisely coincided with the previously demonstrated asymmetric surface domains of sulfogalactoglycerolipid (SGG). Occasionally these SGG-containing, SLIP 1-binding cell surface domains exactly coincided with structural features on the cell surface as detected by differential interference contrast microscopy. These results demonstrate that SLIP 1/SGG interactions could provide an effective intercellular communication network between testicular germ cells within the seminiferous tubule.

  17. Solid-binding Proteins for Modification of Inorganic Substrates

    NASA Astrophysics Data System (ADS)

    Coyle, Brandon Laurence

    Robust and simple strategies to directly functionalize graphene- and diamond-based nanostructures with proteins are of considerable interest for biologically driven manufacturing, biosensing and bioimaging. In this work, we identify a new set of carbon binding peptides that vary in overall hydrophobicity and charge, and engineer two of these sequences (Car9 and Car15) within the framework of various proteins to exploit their binding ability. In addition, we conducted a detailed analysis of the mechanisms that underpin the interaction of the fusion proteins with carbon and silicon surfaces. Through these insights, we were able to develop proteins suitable for dispersing graphene flakes and carbon nanotubes in aqueous solutions, while retaining protein activity. Additionally, our investigation into the mechanisms of adhesion for our carbon binding peptides inspired a cheap, disposable protein purification system that is more than 10x cheaper than commonly used His-tag protein purification. Our results emphasize the importance of understanding both bulk and molecular recognition events when exploiting the adhesive properties of solid-binding peptides and proteins in technological applications.

  18. Theoretical studies of binding of mannose-binding protein to monosaccharides

    NASA Astrophysics Data System (ADS)

    Aida-Hyugaji, Sachiko; Takano, Keiko; Takada, Toshikazu; Hosoya, Haruo; Kojima, Naoya; Mizuochi, Tsuguo; Inoue, Yasushi

    2004-11-01

    Binding properties of mannose-binding protein (MBP) to monosaccharides are discussed based on ab initio molecular orbital calculations for cluster models constructed. The calculated binding energies indicate that MBP has an affinity for N-acetyl- D-glucosamine, D-mannose, L-fucose, and D-glucose rather than D-galactose and N-acetyl- D-galactosamine, which is consistent with the biochemical experimental results. Electrostatic potential surfaces at the binding site of four monosaccharides having binding properties matched well with that of MBP. A vacant frontier orbital was found to be localized around the binding site of MBP, suggesting that MBP-monosaccharide interaction may occur through electrostatic and orbital interactions.

  19. Cholesterol-binding viral proteins in virus entry and morphogenesis.

    PubMed

    Schroeder, Cornelia

    2010-01-01

    Up to now less than a handful of viral cholesterol-binding proteins have been characterized, in HIV, influenza virus and Semliki Forest virus. These are proteins with roles in virus entry or morphogenesis. In the case of the HIV fusion protein gp41 cholesterol binding is attributed to a cholesterol recognition consensus (CRAC) motif in a flexible domain of the ectodomain preceding the trans-membrane segment. This specific CRAC sequence mediates gp41 binding to a cholesterol affinity column. Mutations in this motif arrest virus fusion at the hemifusion stage and modify the ability of the isolated CRAC peptide to induce segregation of cholesterol in artificial membranes.Influenza A virus M2 protein co-purifies with cholesterol. Its proton translocation activity, responsible for virus uncoating, is not cholesterol-dependent, and the transmembrane channel appears too short for integral raft insertion. Cholesterol binding may be mediated by CRAC motifs in the flexible post-TM domain, which harbours three determinants of binding to membrane rafts. Mutation of the CRAC motif of the WSN strain attenuates virulence for mice. Its affinity to the raft-non-raft interface is predicted to target M2 protein to the periphery of lipid raft microdomains, the sites of virus assembly. Its influence on the morphology of budding virus implicates M2 as factor in virus fission at the raft boundary. Moreover, M2 is an essential factor in sorting the segmented genome into virus particles, indicating that M2 also has a role in priming the outgrowth of virus buds.SFV E1 protein is the first viral type-II fusion protein demonstrated to directly bind cholesterol when the fusion peptide loop locks into the target membrane. Cholesterol binding is modulated by another, proximal loop, which is also important during virus budding and as a host range determinant, as shown by mutational studies. PMID:20213541

  20. Studying the protein expression in human B lymphoblastoid cells exposed to 1.8-GHz (GSM) radiofrequency radiation (RFR) with protein microarray

    SciTech Connect

    Zhijian, Chen; Institute of Environmental Health, Medical College, Zhejiang University, Hangzhou 310058, Zhejiang ; Xiaoxue, Li; Wei, Zheng; Yezhen, Lu; Jianlin, Lou; Deqiang, Lu; Shijie, Chen; Lifen, Jin; Jiliang, He

    2013-03-29

    Highlights: ► Protein microarray shows the differential expression of 27 proteins induced by RFR. ► RPA32 related to DNA repair is down-regulated in Western blot. ► p73 related to cell genome stability and apoptosis is up-regulated in Western blot. -- Abstract: In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P < 0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P < 0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P < 0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis.

  1. Detergent activation of the binding protein in the folate radioassay

    SciTech Connect

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with ..beta..-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to ..beta..-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants (lipids/detergents).

  2. DNA-binding proteins in plant mitochondria: implications for transcription.

    PubMed

    Gualberto, José M; Kühn, Kristina

    2014-11-01

    The structural complexity of plant mitochondrial genomes correlates with the variety of single-strand DNA-binding proteins found in plant mitochondria. Most of these are plant-specific and have roles in homologous recombination and genome maintenance. Mitochondrial nucleoids thus differ fundamentally between plants and yeast or animals, where the principal nucleoid protein is a DNA-packaging protein that binds double-stranded DNA. Major transcriptional cofactors identified in mitochondria of non-plant species are also seemingly absent from plants. This article reviews current knowledge on plant mitochondrial DNA-binding proteins and discusses that those may affect the accessibility and conformation of transcription start sites, thus functioning as transcriptional modulators without being dedicated transcription factors. PMID:24561574

  3. Liver takes up retinol-binding protein from plasma

    SciTech Connect

    Gjoen, T.; Bjerkelund, T.; Blomhoff, H.K.; Norum, K.R.; Berg, T.; Blomhoff, R.

    1987-08-15

    Retinol is transported in plasma bound to a specific transport protein, retinol-binding protein. We prepared /sup 125/I-tyramine cellobiose-labeled rat retinol-binding protein and studied its tissue uptake 1, 5, and 24 h after intravenous injection into rats. The liver was the organ containing most radioactivity at all time points studied. After 5 and 24 h, 30 and 22% of the injected dose were recovered in liver, respectively. After separating the liver into parenchymal and nonparenchymal cells in the 5-h group, we found that both cell fractions contained approximately the same amount of radioactivity (per gram of liver). Most of the retinol-binding protein radioactivity in the nonparenchymal cell fraction was in the stellate cells. The implication of these results for a possible transfer mechanism for retinol between parenchymal and stellate cells is discussed.

  4. Tissue microarray analysis reveals a tight correlation between protein expression pattern and progression of esophageal squamous cell carcinoma

    PubMed Central

    Xue, Li-yan; Hu, Nan; Song, Yong-mei; Zou, Shuang-mei; Shou, Jian-zhong; Qian, Lu-xia; Ren, Li-qun; Lin, Dong-mei; Tong, Tong; He, Zu-gen; Zhan, Qi-min; Taylor, Philip R; Lu, Ning

    2006-01-01

    Background The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. Methods Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. Results In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC. Conclusion Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC. PMID:17187659

  5. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins

    PubMed Central

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements. PMID:21087992

  6. PRBP: Prediction of RNA-Binding Proteins Using a Random Forest Algorithm Combined with an RNA-Binding Residue Predictor.

    PubMed

    Ma, Xin; Guo, Jing; Xiao, Ke; Sun, Xiao

    2015-01-01

    The prediction of RNA-binding proteins is an incredibly challenging problem in computational biology. Although great progress has been made using various machine learning approaches with numerous features, the problem is still far from being solved. In this study, we attempt to predict RNA-binding proteins directly from amino acid sequences. A novel approach, PRBP predicts RNA-binding proteins using the information of predicted RNA-binding residues in conjunction with a random forest based method. For a given protein, we first predict its RNA-binding residues and then judge whether the protein binds RNA or not based on information from that prediction. If the protein cannot be identified by the information associated with its predicted RNA-binding residues, then a novel random forest predictor is used to determine if the query protein is a RNA-binding protein. We incorporated features of evolutionary information combined with physicochemical features (EIPP) and amino acid composition feature to establish the random forest predictor. Feature analysis showed that EIPP contributed the most to the prediction of RNA-binding proteins. The results also showed that the information from the RNA-binding residue prediction improved the overall performance of our RNA-binding protein prediction. It is anticipated that the PRBP method will become a useful tool for identifying RNA-binding proteins. A PRBP Web server implementation is freely available at http://www.cbi.seu.edu.cn/PRBP/. PMID:26671809

  7. The RNA-binding protein repertoire of Arabidopsis thaliana.

    PubMed

    Marondedze, Claudius; Thomas, Ludivine; Serrano, Natalia L; Lilley, Kathryn S; Gehring, Chris

    2016-01-01

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category 'RNA-binding', have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses. PMID:27405932

  8. Mercury-binding proteins from the marine mussel, Mytilus edulis

    SciTech Connect

    Roesijadi, G.

    1986-03-01

    The marine mussel, Mytilus edulis, possesses low molecular weight, metal-binding proteins which can be induced by and, in turn, bind mercury when individuals are exposed to low, but elevated concentrations of mercury as HgCl/sub 2/. Induction of the proteins by exposure of mussels to copper, cadmium, or mercury is associated with enhanced tolerance to mercury toxicity. Mercury-binding proteins isolated from gills of mussels occur as two molecular weight variants of about 20-25 and 10-12 kdaltons, respectively, on Sephadex G-75. These have been designated as HgBP/sub 20/ and HgBP/sub 10/ following the nomenclature used for cadmium-binding proteins. HgBP/sub 20/ represents the primary mercury-binding species. Separation of HgBP/sub 20/ by anion-exchange high-performance liquid chromatography resulted in the resolution of six peaks, indicating a more complex situation than was evident from DEAE-cellulose separations. Although not completely purified, these also contain cysteine- and glycine-rich proteins.

  9. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    SciTech Connect

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-02-10

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the ..cap alpha.. subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single ..beta.. subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the ..cap alpha.. subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub s..cap alpha../ relative to G/sub ichemically bond/ and G/sub ochemically bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with (/sup 125/I)protein. Immunohistochemical studies using an antiserum against the ..beta.. subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the ..cap alpha.. subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium.

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

    PubMed

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

    2010-11-01

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

  11. Evaluation of silica nanoparticle binding to major human blood proteins

    NASA Astrophysics Data System (ADS)

    Hata, Katsutomo; Higashisaka, Kazuma; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2014-12-01

    Nanomaterials are used for various biomedical applications because they are often more effective than conventional materials. Recently, however, it has become clear that the protein corona that forms on the surface of nanomaterials when they make contact with biological fluids, such as blood, influences the pharmacokinetics and biological responses induced by the nanomaterials. Therefore, when evaluating nanomaterial safety and efficacy, it is important to analyze the interaction between nanomaterials and proteins in biological fluids and to evaluate the effects of the protein corona. Here, we evaluated the interaction of silica nanoparticles, a commonly used nanomaterial, with the human blood proteins albumin, transferrin, fibrinogen, and IgG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the amount of albumin, transferrin, and IgG binding to the silica particles increased as the particle size decreased under conditions where the silica particle mass remained the same. However, under conditions in which the specific surface area remained constant, there were no differences in the binding of human plasma proteins to the silica particles tested, suggesting that the binding of silica particles with human plasma proteins is dependent on the specific surface area of the silica particles. Furthermore, the amount of albumin, transferrin, and IgG binding to silica nanoparticles with a diameter of 70 nm (nSP70) and a functional amino group was lower than that with unmodified nSP70, although there was no difference in the binding between nSP70 with the surface modification of a carboxyl functional group and nSP70. These results suggest that the characteristics of nanomaterials are important for binding with human blood proteins; this information may contribute to the development of safe and effective nanomaterials.

  12. Building accurate sequence-to-affinity models from high-throughput in vitro protein-DNA binding data using FeatureREDUCE.

    PubMed

    Riley, Todd R; Lazarovici, Allan; Mann, Richard S; Bussemaker, Harmen J

    2015-01-01

    Transcription factors are crucial regulators of gene expression. Accurate quantitative definition of their intrinsic DNA binding preferences is critical to understanding their biological function. High-throughput in vitro technology has recently been used to deeply probe the DNA binding specificity of hundreds of eukaryotic transcription factors, yet algorithms for analyzing such data have not yet fully matured. Here, we present a general framework (FeatureREDUCE) for building sequence-to-affinity models based on a biophysically interpretable and extensible model of protein-DNA interaction that can account for dependencies between nucleotides within the binding interface or multiple modes of binding. When training on protein binding microarray (PBM) data, we use robust regression and modeling of technology-specific biases to infer specificity models of unprecedented accuracy and precision. We provide quantitative validation of our results by comparing to gold-standard data when available. PMID:26701911

  13. Protein D of Haemophilus influenzae is not a universal immunoglobulin D-binding protein.

    PubMed Central

    Sasaki, K; Munson, R S

    1993-01-01

    Haemophilus influenzae type b and nontypeable H. influenzae have been reported to bind human immunoglobulin D (IgD). IgD myeloma sera from five patients were tested for the ability of IgD to bind to H. influenzae. Serotype b strains bound human IgD in four of the five sera tested. IgD in the fifth serum bound strongly to type b strain MinnA but poorly to other type b strains. Additionally, IgD binding was not observed when nontypeable strains were tested. The gene for protein D, the putative IgD-binding protein, was cloned from the IgD-binding H. influenzae type b strain MinnA and expressed in Escherichia coli. IgD binding to E. coli expressing protein D was not demonstrable. Recombinant protein D was purified, and antisera were generated in rabbits. Using these rabbit sera, we detected protein D in nontypeable as well as serotype b strains by Western blotting (immunoblotting). In contrast, IgD myeloma protein 4490, which was previously reported to bind to protein D by Ruan and coworkers (M. Ruan, M. Akkoyunlu, A. Grubb, and A. Forsgren, J. Immunol. 145:3379-3384), bound strongly to both type b and nontypeable H. influenzae as well as to E. coli expressing protein D. Thus, IgD binding is a general property of H. influenzae type b strains but not a general property of nontypeable strains, although both type b and nontypeable strains produce protein D. With the exception of IgD myeloma protein 4490 binding, we have no evidence for a role of protein D in IgD binding to H. influenzae. Images PMID:8514409

  14. Metal binding proteins, recombinant host cells and methods

    DOEpatents

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  15. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    PubMed

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein. PMID:24878641

  16. Lactation-induced cadmium-binding proteins

    SciTech Connect

    Bhattacharyya, M.H.; Solaiman, D.; Garvey, J.S.; Miyazaki, W.Y.

    1987-01-01

    Previously we have demonstrated an increase during midlactation in /sup 109/Cd adsorption and increased retention by the duodenum, kidney, and mammary tissue of mouse dams receiving environmental levels of cadmium//sup 109/Cd via drinking water, with little change in /sup 109/Cd retention in liver and jejunum compared to nonpregnant controls. Results are reported here of a study of cadmium deposition during midlactation as associated with induction of metallothionein (MT). A cadmium/hemoglobin (Cd/Hb) assay and radioimmunoassay for MT which measures heat-stable cadmium binding capacity in tissues was used to determine MT concentrations in fractions of kidney, liver, duodenum, and jejunum from female mice. Both assays demonstrated clear lactation-induced increases in MT concentrations in liver, kidney, and duodenum, with MT concentrations falling rapidly to control levels after weaning. 4 refs., 1 tab.

  17. Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis

    PubMed Central

    Hu, Juan; Kim, Joonyul; Easley, Christopher J.

    2015-01-01

    Effective aptamer-based protein assays require coupling to a quantitative reporter of aptamer-protein binding. Typically, this involves a direct optical or electrochemical readout of DNA hybridization or an amplification step coupled to the readout. However, method development is often hampered by the multiplicity of aptamer-target binding mechanisms, which can interfere with the hybridization step. As a simpler and more generalizable readout of aptamer-protein binding, we report that thermofluorimetric analysis (TFA) can be used to quantitatively assay protein levels. Sub-nanomolar detection (0.74 nM) of platelet-derived growth factor (PDGF) with its corresponding aptamer is shown as a test case. In the presence of various DNA intercalating dyes, protein-bound aptamers exhibit a change in fluorescence intensity compared to the intercalated, unbound aptamer. This allows thermal resolution of bound and unbound aptamers using fluorescence melting analysis (−dF/dT curves). Remarkably, the homogeneous optical method allows subtraction of autofluorescence in human serum, giving PDGF detection limits of 1.8 and 10.7 nM in serum diluted 1:7 and 1:3, respectively. We have thus demonstrated that bound and unbound aptamers can be thermally resolved in a homogeneous format using a simple qPCR instrument—even in human serum. The simplicity of this approach provides an important step toward a robust, generalizable readout of aptamer-protein binding. PMID:26366207

  18. AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

    PubMed Central

    Shen, W F; Montgomery, J C; Rozenfeld, S; Moskow, J J; Lawrence, H J; Buchberg, A M; Largman, C

    1997-01-01

    Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets. PMID:9343407

  19. AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

    PubMed

    Shen, W F; Montgomery, J C; Rozenfeld, S; Moskow, J J; Lawrence, H J; Buchberg, A M; Largman, C

    1997-11-01

    Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets. PMID:9343407

  20. The liver fatty acid binding protein--comparison of cavity properties of intracellular lipid-binding proteins.

    PubMed

    Thompson, J; Ory, J; Reese-Wagoner, A; Banaszak, L

    1999-02-01

    The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound. PMID:10331654

  1. RNA binding proteins in neurodegeneration: Seq and you shall receive

    PubMed Central

    Nussbacher, Julia K.; Batra, Ranjan; Lagier-Tourenne, Clotilde; Yeo, Gene W.

    2015-01-01

    As critical players in gene regulation, RNA binding proteins are taking center stage in our understanding of cellular function and disease. In our era of bench-top sequencers and unprecedented computational power, biological questions can be addressed in a systematic, genome-wide manner. Development of high-throughput sequencing methodologies provides unparalleled potential to discover new mechanisms of disease-associated perturbations of RNA homeostasis. Complementary to candidate single-gene studies, these innovative technologies may elicit the discovery of unexpected mechanisms, and allow us to determine the widespread influence of the multifunctional RNA binding proteins on their targets. As disruption of RNA processing is increasingly implicated in neurological diseases, these approaches will continue to provide insights into the roles of RNA binding proteins in disease pathogenesis. PMID:25765321

  2. Escherchia coli ribose binding protein based bioreporters revisited

    PubMed Central

    Reimer, Artur; Yagur-Kroll, Sharon; Belkin, Shimshon; Roy, Shantanu; van der Meer, Jan Roelof

    2014-01-01

    Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT cause induction of the E. coli reporter system. PMID:25005019

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

    PubMed

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

    2015-08-18

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

  4. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    PubMed

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-01

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner. PMID:27198220

  5. Fluorescent vesicles for signal amplification in reverse phase protein microarray assays.

    PubMed

    Bally, Marta; Syed, Shahida; Binkert, Andreas; Kauffmann, Ekkehard; Ehrat, Markus; Vörös, Janos

    2011-09-15

    Developments in microarray technology promise to lead to great advancements in the biomedical and biological field. However, implementation of these analytical tools often relies on signal amplification strategies that are essential to reach the sensitivity levels required for a variety of biological applications. This is true especially for reverse phase arrays where a complex biological sample is directly immobilized on the chip. We present a simple and generic method for signal amplification based on the use of antibody-tagged fluorescent vesicles as labels for signal generation. To assess the gain in assay sensitivity, we performed a model assay for the detection of rabbit immunoglobulin G (IgG) and compared the limit of detection (LOD) of the vesicle assay with the LOD of a conventional assay performed with fluorescent reporter molecules. We evaluated the improvements for two fluorescence-based transduction setups: a high-sensitivity microarray reader (ZeptoREADER) and a conventional confocal scanner. In all cases, our strategy led to an increase in sensitivity. However, gain in sensitivity widely depended on the type of illumination; whereas an approximately 2-fold increase in sensitivity was observed for readout based on evanescent field illumination, the contribution was as high as more than 200-fold for confocal scanning. PMID:21669176

  6. Binding of drugs in milk: the role of casein in milk protein binding.

    PubMed

    Stebler, T; Guentert, T W

    1990-06-01

    Unbound fractions of 14C-labeled diazepam and tenoxicam in skimmed milk of various species (man, horse, goat, cow, sheep, dog, rabbit) with different milk compositions were determined. Furthermore, the protein binding of five 14C-labeled benzodiazepines differing in their lipophilicity (bromazepam, clonazepam, diazepam, flumazenil, and flunitrazepam) were measured in human milk and in artificially prepared solutions of individual milk proteins (lactoferrin, 2.4 g/liter; alpha-lactalbumin, 2.1 g/liter; albumin, 0.4 g/liter; and casein--2.1, 3.4, and 13.3 g/liter). The extent of binding was determined by equilibrium dialysis of protein solution against 1/15 M phosphate buffer, made isocryoscopic with lactose. The results showed that the casein fraction is a major binding component in milk for all tested drugs. The extent of binding of diazepam and tenoxicam in the milk of various species was independent of the whey protein concentration. In human milk the fraction of bromazepam, clonazepam, diazepam, and flunitrazepam bound to casein was higher than that bound to any other of the milk proteins tested. Albumin contributed little to the overall binding of these benzodiazepines, and lactoferrin and alpha-lactalbumin did not account for significant binding. The benzodiazepine antagonist flumazenil showed the lowest overall binding in milk and in casein solution. As the casein concentration is highest in colostral milk and drops during the course of lactation, it is expected that M/P ratios of drugs strongly bound to casein are higher during the first days postpartum than in later phases of lactation. PMID:2367331

  7. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    SciTech Connect

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  8. MutaBind estimates and interprets the effects of sequence variants on protein-protein interactions.

    PubMed

    Li, Minghui; Simonetti, Franco L; Goncearenco, Alexander; Panchenko, Anna R

    2016-07-01

    Proteins engage in highly selective interactions with their macromolecular partners. Sequence variants that alter protein binding affinity may cause significant perturbations or complete abolishment of function, potentially leading to diseases. There exists a persistent need to develop a mechanistic understanding of impacts of variants on proteins. To address this need we introduce a new computational method MutaBind to evaluate the effects of sequence variants and disease mutations on protein interactions and calculate the quantitative changes in binding affinity. The MutaBind method uses molecular mechanics force fields, statistical potentials and fast side-chain optimization algorithms. The MutaBind server maps mutations on a structural protein complex, calculates the associated changes in binding affinity, determines the deleterious effect of a mutation, estimates the confidence of this prediction and produces a mutant structural model for download. MutaBind can be applied to a large number of problems, including determination of potential driver mutations in cancer and other diseases, elucidation of the effects of sequence variants on protein fitness in evolution and protein design. MutaBind is available at http://www.ncbi.nlm.nih.gov/projects/mutabind/. PMID:27150810

  9. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  10. Solvation structure of ice-binding antifreeze proteins

    NASA Astrophysics Data System (ADS)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

    Antifreeze proteins (AFPs) can be found in organisms which survive at subzero temperatures. They were first discovered in polar fishes since the 1950's [1] and have been isolated meanwhile also from insects, plants, and bacteria. While AFPs shift the freezing point of water below the bulk melting point and hence can prevent recrystallization; the effect is non-colligative and there is a pronounced hysteresis between freezing and melting. For many AFPs it is generally accepted that they function through an irreversible binding to the ice-water interface which leads to a piecewise convex growth front with a lower nonequilibrium freezing point due to the Kelvin effect. Recent molecular dynamics simulations of the AFP from Choristoneura fumiferana reveal that the solvation structures of water at ice-binding and non-ice-binding faces of the protein are crucial for understanding how the AFP binds to the ice surface and how it is protected from being overgrown [2]. We use density functional theory of classical fluids in order to assess the microscopic solvent structure in the vicinity of protein faces with different surface properties. With our method, binding energies of different protein faces to the water-ice-interface can be computed efficiently in a simplified model. [1] Y. Yeh and R.E. Feeney, Chem. Rev. 96, 601 (1996). [2] D.R. Nutt and J.C. Smith, J. Am. Chem. Soc. 130, 13066 (2008).

  11. A Model Membrane Protein for Binding Volatile Anesthetics

    PubMed Central

    Ye, Shixin; Strzalka, Joseph; Churbanova, Inna Y.; Zheng, Songyan; Johansson, Jonas S.; Blasie, J. Kent

    2004-01-01

    Earlier work demonstrated that a water-soluble four-helix bundle protein designed with a cavity in its nonpolar core is capable of binding the volatile anesthetic halothane with near-physiological affinity (0.7 mM Kd). To create a more relevant, model membrane protein receptor for studying the physicochemical specificity of anesthetic binding, we have synthesized a new protein that builds on the anesthetic-binding, hydrophilic four-helix bundle and incorporates a hydrophobic domain capable of ion-channel activity, resulting in an amphiphilic four-helix bundle that forms stable monolayers at the air/water interface. The affinity of the cavity within the core of the bundle for volatile anesthetic binding is decreased by a factor of 4–3.1 mM Kd as compared to its water-soluble counterpart. Nevertheless, the absence of the cavity within the otherwise identical amphiphilic peptide significantly decreases its affinity for halothane similar to its water-soluble counterpart. Specular x-ray reflectivity shows that the amphiphilic protein orients vectorially in Langmuir monolayers at higher surface pressure with its long axis perpendicular to the interface, and that it possesses a length consistent with its design. This provides a successful starting template for probing the nature of the anesthetic-peptide interaction, as well as a potential model system in structure/function correlation for understanding the anesthetic binding mechanism. PMID:15465862

  12. Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer.

    PubMed

    Muralidhara, B K; Wittung-Stafshede, Pernilla

    2003-11-11

    Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected. PMID:14596623

  13. Coenzyme Q10-Binding/Transfer Protein Saposin B also Binds gamma-Tocopherol.

    PubMed

    Jin, Guangzhi; Horinouchi, Ryo; Sagawa, Tomofumi; Orimo, Nobutsune; Kubo, Hiroshi; Yoshimura, Shinichi; Fujisawa, Akio; Kashiba, Misato; Yamamoto, Yorihiro

    2008-09-01

    gamma-Tocopherol, the major form of dietary vitamin E, is absorbed in the intestine and is secreted in chylomicrons, which are then transferred to liver lysosomes. Most gamma-tocopherol is transferred to liver microsomes and is catabolized by cytochrome p450. Due to the hydrophobicity of gamma-tocopherol, a binding and transfer protein is plausible, but none have yet been isolated and characterized. We recently found that a ubiquitous cytosolic protein, saposin B, binds and transfers coenzyme Q10 (CoQ10), which is an essential factor for ATP production and an important antioxidant. Here, we report that saposin B also binds gamma-tocopherol, but not alpha-tocopherol, as efficiently as CoQ10 at pH 7.4. At acidic pH, saposin B binds gamma-tocopherol preferentially to CoQ10 and alpha-tocopherol. Furthermore, we confirmed that saposin B selectively binds gamma-tocopherol instead of CoQ10 and alpha-tocopherol at every pH between 5.4 and 8.0 when all three lipids are competing for binding. We detected gamma-tocopherol in human saposin B monoclonal antibody-induced immunoprecipitates from human urine, although the amount of gamma-tocopherol was much smaller than that of CoQ10. These results suggest that saposin B binds and transports gamma-tocopherol in human cells. PMID:18818759

  14. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes. PMID:19754879

  15. Function analysis of proteins encoded by ORFs 1 to 8 of porcine circovirus-like virus P1 by microarray assay

    PubMed Central

    Wen, Libin; Wang, Fengzhi; Zhang, Dan; He, Kongwang

    2015-01-01

    Porcine circovirus-like agent P1 is a newly discovered virus containing a single-strand circular genome. The genome of P1 is a DNA molecule of 648 nucleotides which contains eight open reading frames (ORFs) that probably encode potential proteins or polypeptides. Thus it is very important to clarify these proteins' function. Here we provide the methods and analysis of microarray data in detail to characterize the transcriptome profile of P1 with and without the ORF. The relevant microarray data sets have been deposited in Gene Expression Omnibus (GEO) database under accession number GSE71945. PMID:26697373

  16. Behind the scenes of vitamin D binding protein: more than vitamin D binding.

    PubMed

    Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M

    2015-10-01

    Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein. PMID:26522461

  17. Studying the protein expression in human B lymphoblastoid cells exposed to 1.8-GHz (GSM) radiofrequency radiation (RFR) with protein microarray.

    PubMed

    Zhijian, Chen; Xiaoxue, Li; Wei, Zheng; Yezhen, Lu; Jianlin, Lou; Deqiang, Lu; Shijie, Chen; Lifen, Jin; Jiliang, He

    2013-03-29

    In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P<0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P<0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P<0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis. PMID:23454122

  18. Structure-based protocol for identifying mutations that enhance protein-protein binding affinities.

    PubMed

    Sammond, Deanne W; Eletr, Ziad M; Purbeck, Carrie; Kimple, Randall J; Siderovski, David P; Kuhlman, Brian

    2007-08-31

    The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: (1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, (2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild-type complex and (3) the mutation should not be predicted to significantly destabilize the monomers. The performance of the computational protocol was experimentally tested on two separate protein complexes; Galpha(i1) from the heterotrimeric G-protein system bound to the RGS14 GoLoco motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. Twelve single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. Nine of the 12 mutations successfully increased binding affinity with five of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the eight mutations identified, five were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities. PMID:17603074

  19. Light-dependent GTP-binding proteins in squid photoreceptors.

    PubMed Central

    Robinson, P R; Wood, S F; Szuts, E Z; Fein, A; Hamm, H E; Lisman, J E

    1990-01-01

    Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2124806

  20. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    PubMed

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP. PMID:25801059

  1. Regioselective covalent immobilization of recombinant antibody-binding proteins A, G, and L for construction of antibody arrays.

    PubMed

    Seo, Jin-soo; Lee, Sungwon; Poulter, C Dale

    2013-06-19

    Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody-binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody-binding proteins are produced in Escherichia coli, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 °C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies. PMID:23746333

  2. Binding-regulated click ligation for selective detection of proteins.

    PubMed

    Cao, Ya; Han, Peng; Wang, Zhuxin; Chen, Weiwei; Shu, Yongqian; Xiang, Yang

    2016-04-15

    Herein, a binding-regulated click ligation (BRCL) strategy for endowing selective detection of proteins is developed with the incorporation of small-molecule ligand and clickable DNA probes. The fundamental principle underlying the strategy is the regulating capability of specific protein-ligand binding against the ligation between clickable DNA probes, which could efficiently combine the detection of particular protein with enormous DNA-based sensing technologies. In this work, the feasibly of the BRCL strategy is first verified through agarose gel electrophoresis and electrochemical impedance spectroscopy measurements, and then confirmed by transferring it to a nanomaterial-assisted fluorescence assay. Significantly, the BRCL strategy-based assay is able to respond to target protein with desirable selectivity, attributing to the specific recognition between small-molecule ligand and its target. Further experiments validate the general applicability of the sensing method by tailoring the ligand toward different proteins (i.e., avidin and folate receptor), and demonstrate its usability in complex biological samples. To our knowledge, this work pioneers the practice of click chemistry in probing specific small-molecule ligand-protein binding, and therefore may pave a new way for selective detection of proteins. PMID:26599478

  3. Quantitative analysis of pheromone-binding protein specificity

    PubMed Central

    Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed. PMID:23121132

  4. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  5. Fast prediction and visualization of protein binding pockets with PASS.

    PubMed

    Brady, G P; Stouten, P F

    2000-05-01

    PASS (Putative Active Sites with Spheres) is a simple computational tool that uses geometry to characterize regions of buried volume in proteins and to identify positions likely to represent binding sites based upon the size, shape, and burial extent of these volumes. Its utility as a predictive tool for binding site identification is tested by predicting known binding sites of proteins in the PDB using both complexed macromolecules and their corresponding apoprotein structures. The results indicate that PASS can serve as a front-end to fast docking. The main utility of PASS lies in the fact that it can analyze a moderate-size protein (approximately 30 kDa) in under 20 s, which makes it suitable for interactive molecular modeling, protein database analysis, and aggressive virtual screening efforts. As a modeling tool, PASS (i) rapidly identifies favorable regions of the protein surface, (ii) simplifies visualization of residues modulating binding in these regions, and (iii) provides a means of directly visualizing buried volume, which is often inferred indirectly from curvature in a surface representation. PASS produces output in the form of standard PDB files, which are suitable for any modeling package, and provides script files to simplify visualization in Cerius2, InsightII, MOE, Quanta, RasMol, and Sybyl. PASS is freely available to all. PMID:10815774

  6. Drug-drug plasma protein binding interactions of ivacaftor.

    PubMed

    Schneider, Elena K; Huang, Johnny X; Carbone, Vincenzo; Baker, Mark; Azad, Mohammad A K; Cooper, Matthew A; Li, Jian; Velkov, Tony

    2015-06-01

    Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. PMID:25707701

  7. The RNA-binding protein repertoire of Arabidopsis thaliana

    PubMed Central

    Marondedze, Claudius; Thomas, Ludivine; Serrano, Natalia L.; Lilley, Kathryn S.; Gehring, Chris

    2016-01-01

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses. PMID:27405932

  8. Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein.

    PubMed Central

    Larrick, J W; Hirata, M; Balint, R F; Lee, J; Zhong, J; Wright, S C

    1995-01-01

    CAP18 (18-kDa cationic antimicrobial protein) is a protein originally identified and purified from rabbit leukocytes on the basis of its capacity to bind and inhibit various activities of lipopolysaccharide (LPS). Here we report the cloning of human CAP18 and characterize the anti-LPS activity of the C-terminal fragment. Oligonucleotide probes designed from the rabbit CAP18 cDNA were used to identify human CAP18 from a bone marrow cDNA library. The cDNA encodes a protein composed of a 30-amino-acid signal peptide, a 103-amino-acid N-terminal domain of unknown function, and a C-terminal domain of 37 amino acids homologous to the LPS-binding antimicrobial domain of rabbit CAP18, designated CAP18(104-140). A human CAP18-specific antiserum was generated by using CAP18 expressed as a fusion protein with the maltose-binding protein. Western blots (immunoblots) with this antiserum showed specific expression of human CAP18 in granulocytes. Synthetic human CAP18(104-140) and a more active truncated fragment, CAP18(104-135), were shown to (i) bind to erythrocytes coated with diverse strains of LPS, (ii) inhibit LPS-induced release of nitric oxide from macrophages, (iii) inhibit LPS-induced generation of tissue factor, and (iv) protect mice from LPS lethality. CAP18(104-140) may have therapeutic utility for conditions associated with elevated concentrations of LPS. PMID:7890387

  9. Stable, ligand-doped, poly(bis-SorbPC) lipid bilayer arrays for protein binding and detection.

    PubMed

    Joubert, James R; Smith, Kathryn A; Johnson, Erin; Keogh, John P; Wysocki, Vicki H; Gale, Bruce K; Conboy, John C; Saavedra, S Scott

    2009-06-01

    A continuous-flow microspotter was used to generate planar arrays of stabilized bilayers composed of the polymerizable lipid bis-SorbPC and dopant lipids bearing ligands for proteins. Fluorescence microscopy was used to determine the uniformity of the bilayers and to detect protein binding. After UV-initiated polymerization, poly(lipid) bilayer microarrays were air-stable. Cholera toxin subunit b (CTb) bound to an array of poly(lipid) bilayers doped with GM(1), and the extent of binding was correlated to the mole percentage of GM(1) in each spot. A poly(lipid) bilayer array composed of spots doped with GM(1) and spots doped with biotin-DOPE specifically bound CTb and streptavidin to the respective spots from a dissolved mixture of the two proteins. Poly(bis-SorbPC)/GM(1) arrays retained specific CTb binding capacity after multiple regenerations with a protein denaturing solution and also after exposure to air. In addition, these arrays are stable in vacuum, which allows the use of MALDI-TOF mass spectrometry to detect specifically bound CTb. This work demonstrates the considerable potential of poly(lipid) bilayer arrays for high-throughput binding assays and lipidomics studies. PMID:20355927

  10. Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts

    PubMed Central

    2010-01-01

    Background Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly. Results Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested. Conclusion The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the in

  11. Chemokine binding proteins: An immunomodulatory strategy going viral.

    PubMed

    González-Motos, Víctor; Kropp, Kai A; Viejo-Borbolla, Abel

    2016-08-01

    Chemokines are chemotactic cytokines whose main function is to direct cell migration. The chemokine network is highly complex and its deregulation is linked to several diseases including immunopathology, cancer and chronic pain. Chemokines also play essential roles in the antiviral immune response. Viruses have therefore developed several counter strategies to modulate chemokine activity. One of these is the expression of type I transmembrane or secreted proteins with the ability to bind chemokines and modulate their activity. These proteins, termed viral chemokine binding proteins (vCKBP), do not share sequence homology with host proteins and are immunomodulatory in vivo. In this review we describe the discovery and characterization of vCKBP, explain their role in the context of infection in vivo and discuss relevant novel findings. PMID:26987612

  12. Protein Binding for Detection of Small Changes on Nanoparticle Surface

    PubMed Central

    Zeng, Shang; Huang, Yu-ming M.; Chang, Chia-en A.; Zhong, Wenwan

    2014-01-01

    Protein adsorption on nanoparticles is closely associated with the physicochemical properties of particles, in particular, their surface property. We synthesized two batches of polyacrylic acid-coated nanoparticles under almost identical conditions except for heating duration and found differences in the head-group structure of the polyacrylic acid. The structure change was confirmed by NMR and MS. The two batches of particles had varied binding affinities to a selected group of proteins. Computational work confirmed that the head group of the polymer on the surface of a nanoparticle could directly interact with a protein, and small structural changes in the head group were sufficient to result in a significant difference in the free energy of binding. Our results demonstrate that protein adsorption is so sensitive to the surface property of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand, and should be useful for quick assessment of nanoparticle properties. PMID:24482794

  13. Ag@SiO2-entrapped hydrogel microarray: a new platform for a metal-enhanced fluorescence-based protein assay.

    PubMed

    Jang, Eunji; Kim, Minsu; Koh, Won-Gun

    2015-05-21

    We developed a novel protein-based bioassay platform utilizing metal-enhanced fluorescence (MEF), which is a hydrogel microarray entrapping silica-coated silver nanoparticles (Ag@SiO2). As a model system, different concentrations of glucose were detected using a fluorescence method by sequential bienzymatic reaction of hydrogel-entrapped glucose oxidase (GOX) and peroxidase (POD) inside a hydrogel microarray. Microarrays based on poly(ethylene glycol)(PEG) hydrogels were prepared by photopatterning a solution containing PEG diacrylate (PEG-DA), photoinitiator, enzymes, and Ag@SiO2. The resulting hydrogel microarrays were able to entrap both enzymes and Ag@SiO2 without leaching and deactivation problems. The presence of Ag@SiO2 within the hydrogel microarray enhanced the fluorescence signal, and the extent of the enhancement was dependent on the thickness of silica shells and the amount of Ag@SiO2. Optimal MEF effects were achieved when the thickness of the silica shell was 17.5 nm, and 0.5 mg mL(-1) of Ag@SiO2 was incorporated into the assay systems. Compared with the standard hydrogel microarray-based assay performed without Ag@SiO2, more than a 4-fold fluorescence enhancement was observed in a glucose concentration range between 10(-3) mM and 10.0 mM using hydrogel microarray entrapping Ag@SiO2, which led to significant improvements in the sensitivity and the limit of detection (LOD). The hydrogel microarray system presented in this study could be successfully combined with a microfluidic device as an initial step to create an MEF-based micro-total-analysis-system (μ-TAS). PMID:25837891

  14. Drug bioactivation, covalent binding to target proteins and toxicity relevance.

    PubMed

    Zhou, Shufeng; Chan, Eli; Duan, Wei; Huang, Min; Chen, Yu-Zong

    2005-01-01

    A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients

  15. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection

    PubMed Central

    Chiang, Chih-Yuan; Uzoma, Ijeoma; Lane, Douglas J.; Memišević, Vesna; Alem, Farhang; Yao, Kuan; Kota, Krishna P.; Bavari, Sina; Wallqvist, Anders; Hakami, Ramin M.; Panchal, Rekha G.

    2015-01-01

    Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments. PMID:26284031

  16. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection.

    PubMed

    Chiang, Chih-Yuan; Uzoma, Ijeoma; Lane, Douglas J; Memišević, Vesna; Alem, Farhang; Yao, Kuan; Kota, Krishna P; Bavari, Sina; Wallqvist, Anders; Hakami, Ramin M; Panchal, Rekha G

    2015-01-01

    Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments. PMID:26284031

  17. A general approach to visualize protein binding and DNA conformation without protein labelling

    PubMed Central

    Song, Dan; Graham, Thomas G. W.; Loparo, Joseph J.

    2016-01-01

    Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein–DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein–DNA interactions. PMID:26952553

  18. Development and Optimization of a Thrombin Sandwich Aptamer Microarray

    PubMed Central

    Meneghello, Anna; Sosic, Alice; Antognoli, Agnese; Cretaio, Erica; Gatto, Barbara

    2012-01-01

    A sandwich microarray employing two distinct aptamers for human thrombin has been optimized for the detection of subnanomolar concentrations of the protein. The aptamer microarray demonstrates high specificity for thrombin, proving that a two-site binding assay with the TBA1 aptamer as capture layer and the TBA2 aptamer as detection layer can ensure great specificity at times and conditions compatible with standard routine analysis of biological samples. Aptamer microarray sensitivity was evaluated directly by fluorescent analysis employing Cy5-labeled TBA2 and indirectly by the use of TBA2-biotin followed by detection with fluorescent streptavidin. Sub-nanomolar LODs were reached in all cases and in the presence of serum, demonstrating that the optimized aptamer microarray can identify thrombin by a low-cost, sensitive and specific method.

  19. DNA binding proteins that alter nucleic acid flexibility

    NASA Astrophysics Data System (ADS)

    McCauley, Micah; Hardwidge, Philip R.; Maher, L. J., III; Williams, Mark C.

    2007-09-01

    Dual - beam optical tweezers experiments subject single molecules of DNA to high forces (~ 300 pN) with 0.1 pN accuracy, probing the energy and specificity of nucleic acid - ligand structures. Stretching phage λ-DNA reveals an increase in the applied force up to a critical force known as the overstretching transition. In this region, base pairing and stacking are disrupted as double stranded DNA (dsDNA) is melted. Proteins that bind to the double strand will tend to stabilize dsDNA, and melting will occur at higher forces. Proteins that bind to single stranded DNA (ssDNA) destabilize melting, provided that the rate of association is comparable to the pulling rate of the experiment. Many proteins, however, exhibit some affinity for both dsDNA and ssDNA. We describe experiments upon DNA + HMGB2 (box A), a nuclear protein that is believed to facilitate transcription. By characterizing changes in the structure of dsDNA with a polymer model of elasticity, we have determined the equilibrium association constant for HMGB2 to be K ds = 0.15 +/- 0.7 10 9 M -1 for dsDNA binding. Analysis of the melting transition reveals an equilibrium association constant for HMGB2 to ssDNA to be K ss = 0.039 +/- 0.019 10 9 M -1 for ssDNA binding.

  20. Capacitance-modulated transistor detects odorant binding protein chiral interactions.

    PubMed

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters. PMID:25591754

  1. Protein-Ligand Binding Detected by Terahertz Spectroscopy

    NASA Astrophysics Data System (ADS)

    Knab, J.; Chen, J. Y.; Mader, M.; Markelz, A.

    2004-03-01

    Established measures of protein flexibility through the B-factor use time intensive and facility limited techniques such as X-ray crystallography, NMR structure analysis and inelastic neutron scattering. We demonstrate a novel technique that may be used for determination of ligand binding for proteins as well as a measure of protein flexibility. Using the method of terahertz (THz) time domain spectroscopy, we measured the far infrared dielectric response as a function of the binding of N (1-4)-acetylglucosamine (NAG) to hen egg white lysozyme (HEWL). Vibrational modes associated with tertiary structure conformational motions lay in the THz frequency range. The THz dielectric response reflects the density and amplitude of these normal modes through dipole coupling. Transmission measurements on thin films show that while there is no change in the real part of the refractive index as a function of binding, there is a decrease in the absorbance for the HEWL+NAG thin films relative to HEWL films. This decrease can be attributed to a reduction in the flexibility of the protein with binding. These results are compared to calculated absorbance spectra.

  2. Methods of use of cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1997-09-23

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  3. Methods of use of cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1997-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  4. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    NASA Astrophysics Data System (ADS)

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

  5. Microarray Analysis in Glioblastomas.

    PubMed

    Bhawe, Kaumudi M; Aghi, Manish K

    2016-01-01

    Microarray analysis in glioblastomas is done using either cell lines or patient samples as starting material. A survey of the current literature points to transcript-based microarrays and immunohistochemistry (IHC)-based tissue microarrays as being the preferred methods of choice in cancers of neurological origin. Microarray analysis may be carried out for various purposes including the following: i. To correlate gene expression signatures of glioblastoma cell lines or tumors with response to chemotherapy (DeLay et al., Clin Cancer Res 18(10):2930-2942, 2012). ii. To correlate gene expression patterns with biological features like proliferation or invasiveness of the glioblastoma cells (Jiang et al., PLoS One 8(6):e66008, 2013). iii. To discover new tumor classificatory systems based on gene expression signature, and to correlate therapeutic response and prognosis with these signatures (Huse et al., Annu Rev Med 64(1):59-70, 2013; Verhaak et al., Cancer Cell 17(1):98-110, 2010). While investigators can sometimes use archived tumor gene expression data available from repositories such as the NCBI Gene Expression Omnibus to answer their questions, new arrays must often be run to adequately answer specific questions. Here, we provide a detailed description of microarray methodologies, how to select the appropriate methodology for a given question, and analytical strategies that can be used. Experimental methodology for protein microarrays is outside the scope of this chapter, but basic sample preparation techniques for transcript-based microarrays are included here. PMID:26113463

  6. Multiple Binding Poses in the Hydrophobic Cavity of Bee Odorant Binding Protein AmelOBP14.

    PubMed

    Pechlaner, Maria; Oostenbrink, Chris

    2015-12-28

    In the first step of olfaction, odorants are bound and solubilized by small globular odorant binding proteins (OBPs) which shuttle them to the membrane of a sensory neuron. Low ligand affinity and selectivity at this step enable the recognition of a wide range of chemicals. Honey bee Apis mellifera's OBP14 (AmelOBP14) binds different plant odorants in a largely hydrophobic cavity. In long molecular dynamics simulations in the presence and absence of ligand eugenol, we observe a highly dynamic C-terminal region which forms one side of the ligand-binding cavity, and the ligand drifts away from its crystallized orientation. Hamiltonian replica exchange simulations, allowing exchanges of conformations sampled by the real ligand with those sampled by a noninteracting dummy molecule and several intermediates, suggest an alternative, quite different ligand pose which is adopted immediately and which is stable in long simulations. Thermodynamic integration yields binding free energies which are in reasonable agreement with experimental data. PMID:26633245

  7. Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4)

    PubMed Central

    González, Javier M.; Fisher, S. Zoë

    2015-01-01

    Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments. PMID:25664790

  8. The neuronal calcium sensor family of Ca2+-binding proteins.

    PubMed Central

    Burgoyne, R D; Weiss, J L

    2001-01-01

    Ca(2+) plays a central role in the function of neurons as the trigger for neurotransmitter release, and many aspects of neuronal activity, from rapid modulation to changes in gene expression, are controlled by Ca(2+). These actions of Ca(2+) must be mediated by Ca(2+)-binding proteins, including calmodulin, which is involved in Ca(2+) regulation, not only in neurons, but in most other cell types. A large number of other EF-hand-containing Ca(2+)-binding proteins are known. One family of these, the neuronal calcium sensor (NCS) proteins, has a restricted expression in retinal photoreceptors or neurons and neuroendocrine cells, suggesting that they have specialized roles in these cell types. Two members of the family (recoverin and guanylate cyclase-activating protein) have established roles in the regulation of phototransduction. Despite close sequence similarities, the NCS proteins have distinct neuronal distributions, suggesting that they have different functions. Recent work has begun to demonstrate the physiological roles of members of this protein family. These include roles in the modulation of neurotransmitter release, control of cyclic nucleotide metabolism, biosynthesis of polyphosphoinositides, regulation of gene expression and in the direct regulation of ion channels. In the present review we describe the known sequences and structures of the NCS proteins, information on their interactions with target proteins and current knowledge about their cellular and physiological functions. PMID:11115393

  9. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation.

    PubMed

    Weidmann, Chase A; Raynard, Nathan A; Blewett, Nathan H; Van Etten, Jamie; Goldstrohm, Aaron C

    2014-08-01

    PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation. PMID:24942623

  10. Studies on fatty acid-binding proteins. The diurnal variation shown by rat liver fatty acid-binding protein.

    PubMed Central

    Wilkinson, T C; Wilton, D C

    1987-01-01

    The concentration of fatty acid-binding protein in rat liver was examined by SDS/polyacrylamide-gel electrophoresis, by Western blotting and by quantifying the fluorescence enhancement achieved on the binding of the fluorescent probe 11-(dansylamino)undecanoic acid. A 2-3-fold increase in the concentration of this protein produced by treatment of rats with the peroxisome proliferator tiadenol was readily detected; however, only a small variation in the concentration of the protein due to a diurnal rhythm was observed. This result contradicts the 7-10-fold variation previously reported for this protein [Hargis, Olson, Clarke & Dempsey (1986) J. Biol. Chem. 261, 1988-1991]. Images Fig. 1. Fig. 3. PMID:3593284

  11. RNA–protein binding interface in the telomerase ribonucleoprotein

    PubMed Central

    Bley, Christopher J.; Qi, Xiaodong; Rand, Dustin P.; Borges, Chad R.; Nelson, Randall W.; Chen, Julian J.-L.

    2011-01-01

    Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR–TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA–protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain. PMID:22123986

  12. Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance.

    PubMed

    Nguyen, Cam Chau; Nakaminami, Kentaro; Matsui, Akihiro; Kobayashi, Shuhei; Kurihara, Yukio; Toyooka, Kiminori; Tanaka, Maho; Seki, Motoaki

    2016-01-01

    Stress granules (SGs), which are formed in the plant cytoplasm under stress conditions, are transient dynamic sites (particles) for mRNA storage. SGs are actively involved in protecting mRNAs from degradation. Oligouridylate binding protein 1b (UBP1b) is a component of SGs. The formation of microscopically visible cytoplasmic foci, referred to as UBP1b SG, was induced by heat treatment in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox). A detailed understanding of the function of UBP1b, however, is still not clear. UBP1b-ox plants displayed increased heat tolerance, relative to control plants, while ubp1b mutants were more sensitive to heat stress than control plants. Microarray analysis identified 117 genes whose expression was heat-inducible and higher in the UBP1b-ox plants. RNA decay analysis was performed using cordycepin, a transcriptional inhibitor. In order to determine if those genes serve as targets of UBP1b, the rate of RNA degradation of a DnaJ heat shock protein and a stress-associated protein (AtSAP3) in UBP1b-ox plants was slower than in control plants; indicating that the mRNAs of these genes were protected within the UBP1b SG granule. Collectively, these data demonstrate that UBP1b plays an integral role in heat stress tolerance in plants. PMID:27379136

  13. Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance

    PubMed Central

    Nguyen, Cam Chau; Nakaminami, Kentaro; Matsui, Akihiro; Kobayashi, Shuhei; Kurihara, Yukio; Toyooka, Kiminori; Tanaka, Maho; Seki, Motoaki

    2016-01-01

    Stress granules (SGs), which are formed in the plant cytoplasm under stress conditions, are transient dynamic sites (particles) for mRNA storage. SGs are actively involved in protecting mRNAs from degradation. Oligouridylate binding protein 1b (UBP1b) is a component of SGs. The formation of microscopically visible cytoplasmic foci, referred to as UBP1b SG, was induced by heat treatment in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox). A detailed understanding of the function of UBP1b, however, is still not clear. UBP1b-ox plants displayed increased heat tolerance, relative to control plants, while ubp1b mutants were more sensitive to heat stress than control plants. Microarray analysis identified 117 genes whose expression was heat-inducible and higher in the UBP1b-ox plants. RNA decay analysis was performed using cordycepin, a transcriptional inhibitor. In order to determine if those genes serve as targets of UBP1b, the rate of RNA degradation of a DnaJ heat shock protein and a stress-associated protein (AtSAP3) in UBP1b-ox plants was slower than in control plants; indicating that the mRNAs of these genes were protected within the UBP1b SG granule. Collectively, these data demonstrate that UBP1b plays an integral role in heat stress tolerance in plants. PMID:27379136

  14. Observation of Protein Structural Vibrational Mode Sensitivity to Ligand Binding

    NASA Astrophysics Data System (ADS)

    Niessen, Katherine; Xu, Mengyang; Snell, Edward; Markelz, Andrea

    2014-03-01

    We report the first measurements of the dependence of large-scale protein intramolecular vibrational modes on ligand binding. These collective vibrational modes in the terahertz (THz) frequency range (5-100 cm-1) are of great interest due to their predicted relation to protein function. Our technique, Crystals Anisotropy Terahertz Microscopy (CATM), allows for room temperature, table-top measurements of the optically active intramolecular modes. CATM measurements have revealed surprisingly narrowband features. CATM measurements are performed on single crystals of chicken egg-white lysozyme (CEWL) as well as CEWL bound to tri-N-acetylglucosamine (CEWL-3NAG) inhibitor. We find narrow band resonances that dramatically shift with binding. Quasiharmonic calculations are performed on CEWL and CEWL-3NAG proteins with CHARMM using normal mode analysis. The expected CATM response of the crystals is then calculated by summing over all protein orientations within the unit cell. We will compare the CATM measurements with the calculated results and discuss the changes which arise with protein-ligand binding. This work is supported by NSF grant MRI 2 grant DBI2959989.

  15. Cellular Actions of Insulin-Like Growth Factor Binding Proteins

    PubMed Central

    Ferry, R. J.; Katz, L. E. L.; Grimberg, Adda; Cohen, P.; Weinzimer, S. A.

    2014-01-01

    The insulin-like growth factors (IGFs), insulin-like growth factor binding proteins (IGFBPs), and the IGFBP proteases are involved in the regulation of somatic growth and cellular proliferation both in vivo and in vitro. IGFs are potent mitogenic agents whose actions are determined by the availability of free IGFs to interact with the IGF receptors. IGFBPs comprise a family of proteins that bind IGFs with high affinity and specificity and thereby regulate IGF-dependent actions. IGFBPs have recently emerged as IGF-independent regulators of cell growth. Various IGFBP association proteins as well as cleavage of IGFBPs by specific proteases modulate levels of free IGFs and IGFBPs. The ubiquity and complexity of the IGF axis promise exciting discoveries and applications for the future. PMID:10226802

  16. Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

    PubMed

    Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

    2016-12-15

    In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. PMID:27451201

  17. Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research*

    PubMed Central

    Pedersen, Henriette L.; Fangel, Jonatan U.; McCleary, Barry; Ruzanski, Christian; Rydahl, Maja G.; Ralet, Marie-Christine; Farkas, Vladimir; von Schantz, Laura; Marcus, Susan E.; Andersen, Mathias C. F.; Field, Rob; Ohlin, Mats; Knox, J. Paul; Clausen, Mads H.; Willats, William G. T.

    2012-01-01

    Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes. PMID:22988248

  18. Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

    PubMed

    Pleass, R J; Areschoug, T; Lindahl, G; Woof, J M

    2001-03-16

    Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function. PMID:11096107

  19. Polyamine binding to proteins in oat and Petunia protoplasts

    NASA Technical Reports Server (NTRS)

    Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

  20. Polyamine Binding to Proteins in Oat and Petunia Protoplasts 1

    PubMed Central

    Mizrahi, Yosef; Applewhite, Philip B.; Galston, Arthur W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozenthawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein. Images Figure 2 PMID:11537462

  1. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes. PMID:26361975

  2. Single prenyl-binding site on protein prenyl transferases

    PubMed Central

    Desnoyers, Luc; Seabra, Miguel C.

    1998-01-01

    Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions. PMID:9770475

  3. Protein turnover in atrophying muscle: from nutritional intervention to microarray expression analysis

    NASA Technical Reports Server (NTRS)

    Stein, T. Peter; Wade, Charles E.

    2003-01-01

    PURPOSE OF REVIEW: In response to decreased usage, skeletal muscle undergoes adaptive reductive remodeling due to the decrease in tension on the weight bearing components of the musculo-skeletal system. This response occurs with uncomplicated disuse (e.g. bed rest, space flight), as a secondary consequence of several widely prevalent chronic diseases for which activity is reduced (e.g. chronic obstructive pulmonary disease and chronic heart failure) and is part of the aging process. The problem is therefore one of considerable clinical importance. RECENT FINDINGS: The impaired function and exercise intolerance is related more to the associated muscle wasting rather than to the specific organ system primarily impacted by the disease. Progress has continued in describing the use of anabolic drugs and dietary manipulation. The major advance in the field has been: (i) the discovery of the atrogin-1 gene and (ii) the application of microarray expression analysis and proteomics with the objectives of obtaining comprehensive understanding of the pathways changed with disuse atrophy. SUMMARY: Disuse atrophy is a common clinical problem. There is a need for therapeutic interventions that do not involve exercise. A better understanding of the changes, particularly at the molecular level, could indicate hitherto unsuspected sites for nutritional and pharmacological intervention.

  4. Integrative Proteomics and Tissue Microarray Profiling Indicate the Association between Overexpressed Serum Proteins and Non-Small Cell Lung Cancer

    PubMed Central

    Hu, Haichuan; Wang, Rui; Sun, Yihua; Zeng, Rong; Chen, Haiquan

    2012-01-01

    Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC) can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA) and Multiple reaction monitoring (MRM) assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases. PMID:23284758

  5. Microbiota Modulates Behavior and Protein Kinase C mediated cAMP response element-binding protein Signaling

    PubMed Central

    Zeng, Li; Zeng, Benhua; Wang, Haiyang; Li, Bo; Huo, Ran; Zheng, Peng; Zhang, Xiaotong; Du, Xiangyu; Liu, Meiling; Fang, Zheng; Xu, Xuejiao; Zhou, Chanjuan; Chen, Jianjun; Li, Wenxia; Guo, Jing; Wei, Hong; Xie, Peng

    2016-01-01

    Evolutionary pressure drives gut microbiota–host coevolution and results in complex interactions between gut microbiota and neural development; however, the molecular mechanisms by which the microbiota governs host behavior remain obscure. Here, we report that colonization early in life is crucial for the microbiota to modulate brain development and behavior; later colonization or deletion of microbiota cannot completely reverse the behaviors. Microarray analysis revealed an association between absence of gut microbiota and expression in cAMP responding element-binding protein (CREB) regulated genes in the hippocampus. The absence of gut microbiota from birth was shown to be associated with decreased CREB expression, followed by decreases of protein kinase C beta (PRKCB) and AMPA receptors expression, and an increase of phosphorylation CREB (pCREB) expression. Microbiota colonization in adolescence restored CREB and pCREB expression, but did not alter PRKCB and AMPARs expression. The removal of the gut microbiota from SPF mice using antibiotics only reduced pCREB expression. These findings suggest that (i) colonization of the gut microbiota early in life might facilitate neurodevelopment via PKC–CREB signaling and (ii) although GF mice and ABX mice display reduced anxiety-related behaviors, the molecular mechanisms behind this might differ. PMID:27444685

  6. Microbiota Modulates Behavior and Protein Kinase C mediated cAMP response element-binding protein Signaling.

    PubMed

    Zeng, Li; Zeng, Benhua; Wang, Haiyang; Li, Bo; Huo, Ran; Zheng, Peng; Zhang, Xiaotong; Du, Xiangyu; Liu, Meiling; Fang, Zheng; Xu, Xuejiao; Zhou, Chanjuan; Chen, Jianjun; Li, Wenxia; Guo, Jing; Wei, Hong; Xie, Peng

    2016-01-01

    Evolutionary pressure drives gut microbiota-host coevolution and results in complex interactions between gut microbiota and neural development; however, the molecular mechanisms by which the microbiota governs host behavior remain obscure. Here, we report that colonization early in life is crucial for the microbiota to modulate brain development and behavior; later colonization or deletion of microbiota cannot completely reverse the behaviors. Microarray analysis revealed an association between absence of gut microbiota and expression in cAMP responding element-binding protein (CREB) regulated genes in the hippocampus. The absence of gut microbiota from birth was shown to be associated with decreased CREB expression, followed by decreases of protein kinase C beta (PRKCB) and AMPA receptors expression, and an increase of phosphorylation CREB (pCREB) expression. Microbiota colonization in adolescence restored CREB and pCREB expression, but did not alter PRKCB and AMPARs expression. The removal of the gut microbiota from SPF mice using antibiotics only reduced pCREB expression. These findings suggest that (i) colonization of the gut microbiota early in life might facilitate neurodevelopment via PKC-CREB signaling and (ii) although GF mice and ABX mice display reduced anxiety-related behaviors, the molecular mechanisms behind this might differ. PMID:27444685

  7. Relationship between binding affinities to cellular retinoic acid-binding protein and biological potency of a new series of retinoids.

    PubMed

    Sani, B P; Dawson, M I; Hobbs, P D; Chan, R L; Schiff, L J

    1984-01-01

    Binding affinities of a new and unusual series of retinoic acid analogues to cellular retinoic acid-binding protein, a possible mediator of their biological function in the control of differentiation and tumorigenesis, and to serum albumin, their plasma transport protein, were determined. Also, biological activity of these retinoids in the reversal of keratinization in hamster tracheal organ cultures was assessed and compared with their binding affinities. Analogues that possessed high biological activity showed high binding efficiency to cellular retinoic acid-binding protein. Those that were biologically less active were poor binders to the binding protein. Three retinoids, 4657-57, 3920-59, and 4445-75, which showed 90 to 100% binding efficiency of that of retinoic acid for cellular retinoic acid-binding protein expressed high biological activity detectable in the range of 10(-10) M as against 10(-11) M for retinoic acid. The correlation noticed in these two activities not only enhances the confidence in the two assay procedures but also paves the way for design and development of potential chemopreventive agents. No apparent differences were observed in the binding affinities of the retinoids to binding proteins of a normal tissue or a tumor tissue. No correlation existed between the binding affinities of these retinoids to serum albumin and their biological activity. Structure-activity relationships of the retinoids in relation to their binding affinities and biological activities have been discussed. PMID:6317169

  8. RNA binding by the Wilms tumor suppressor zinc finger proteins.

    PubMed Central

    Caricasole, A; Duarte, A; Larsson, S H; Hastie, N D; Little, M; Holmes, G; Todorov, I; Ward, A

    1996-01-01

    The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms. Images Fig. 1 Fig. 2 Fig. 3 PMID:8755514

  9. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    PubMed Central

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand–protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein–ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters. PMID:25591754

  10. Molecular cloning of a human protein that binds to the retinoblastoma protein and chromosomal mapping

    SciTech Connect

    Saijo, M.; Sakai, Y.; Taya, Y.

    1995-06-10

    We have isolated distinct clones for cellular proteins that bind to the retinoblastoma protein by direct screening of cDNA expression libraries using purified pRB as a probe. The total nucleotide sequence of one of these clones, RBQ-3, was determined and found to encode a protein of 66 kDa localized in the nucleus. The RBQ-3 preferentially binds to underphosphory-lated pRB. The region used for binding to this protein was mapped to the E1A-binding pocket B of pRB, which has sequence similarity to the general transcription factor TFIIB. We have mapped the gene to 1q32 using polymerase chain reaction analysis on a human-hamster hybrid cell panel and chromosomal fluorescence in situ hybridization. 64 refs., 7 figs.

  11. Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase

    PubMed Central

    Sosic, Alice; Meneghello, Anna; Cretaio, Erica; Gatto, Barbara

    2011-01-01

    We have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM) and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized. PMID:22163703

  12. Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans

    NASA Astrophysics Data System (ADS)

    Zhao, Nan; Martin, Brigitte E.; Yang, Chun-Kai; Luo, Feng; Wan, Xiu-Feng

    2015-10-01

    Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2-6Galβ, human-origin viruses could bind glycans with Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galβ1-4GlcNAc substructures; Galβ and GlcNAcβ terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2-3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms.

  13. Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans.

    PubMed

    Zhao, Nan; Martin, Brigitte E; Yang, Chun-Kai; Luo, Feng; Wan, Xiu-Feng

    2015-01-01

    Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2-6Galβ, human-origin viruses could bind glycans with Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galβ1-4GlcNAc substructures; Galβ and GlcNAcβ terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2-3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms. PMID:26508590

  14. Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans

    PubMed Central

    Zhao, Nan; Martin, Brigitte E.; Yang, Chun-Kai; Luo, Feng; Wan, Xiu-Feng

    2015-01-01

    Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2–6Galβ, human-origin viruses could bind glycans with Neu5Acα2–8Neu5Acα2–8Neu5Ac and Neu5Gcα2–6Galβ1–4GlcNAc substructures; Galβ and GlcNAcβ terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2–3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms. PMID:26508590

  15. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

    PubMed

    Tottey, Steve; Waldron, Kevin J; Firbank, Susan J; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R; Gray, Joe; Banfield, Mark J; Dennison, Christopher; Robinson, Nigel J

    2008-10-23

    Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+). PMID:18948958

  16. Species differences in the binding kinetics of 25-hydroxyvitamin D3 to vitamin D binding protein.

    PubMed

    Vieth, R; Kessler, M J; Pritzker, K P

    1990-10-01

    The specific binding of 25-hydroxyvitamin D3 to its binding protein was studied in serum of the human, rhesus monkey, cow, horse, and rat. The free fraction of 25-hydroxyvitamin D3 in the rat was 0.34 +/- 0.15 pmol free/nmol total (+/- SD) and this was lower than in any of the other species (p less than 0.01). In the human, the free fraction was 1.5 +/- 0.32 pmol free/nmol total, which was higher than in any of the other species (p less than 0.001). The differences in the free fraction were mainly due to differences in dissociation constant. The relative levels of free 25-hydroxyvitamin D should be taken into account when extrapolating findings about vitamin D metabolism in animals to the human. A technical outcome of this study is that of the species tested, vitamin D binding protein from rat serum is the most suitable as a reagent component for methods used to measure total 25-hydroxyvitamin D by competitive protein binding assay. PMID:2078829

  17. [Penicillin-binding proteins of various strains of Lactobacillus].

    PubMed

    Griaznova, N S; Subbotina, N A; Beliavskaia, I V; Taisova, A S; Afonin, V I; Tiurin, M V; Shenderov, B A; Sazykina, Iu O; Navashin, S M

    1990-02-01

    Sensitivity of different species of Lactobacillus i.e. L. casei, L. plantarum, L. acidophillus, L. buchneri, L. jugurti and others to penicillins and cephalosporins of various generations was studied. Penicillin binding proteins (PBPs) of the Lactobacillus species were specified. It was shown that the number of PBPs depended on the Lactobacillus species. L. casei had the least number of PBPs (4) and L. brevis had the highest number of PBPs (11). Competition of 14C-benzylpenicillin with ampicillin, cefotaxime, ceftizoxime and cefoperazone for binding to separate PBPs in three strains of different Lactobacillus species was investigated. PMID:2110806

  18. Photoaffinity labelling of high affinity dopamine binding proteins

    SciTech Connect

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

    1986-03-01

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

  19. Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity

    PubMed Central

    Korkuć, Paula; Walther, Dirk

    2015-01-01

    To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous

  20. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

  1. Binding of TATA Binding Protein to a Naturally Positioned Nucleosome Is Facilitated by Histone Acetylation

    PubMed Central

    Sewack, Gerald F.; Ellis, Thomas W.; Hansen, Ulla

    2001-01-01

    The TATA sequence of the human, estrogen-responsive pS2 promoter is complexed in vivo with a rotationally and translationally positioned nucleosome (NUC T). Using a chromatin immunoprecipitation assay, we demonstrate that TATA binding protein (TBP) does not detectably interact with this genomic binding site in MCF-7 cells in the absence of transcriptional stimuli. Estrogen stimulation of these cells results in hyperacetylation of both histones H3 and H4 within the pS2 chromatin encompassing NUC T and the TATA sequence. Concurrently, TBP becomes associated with the pS2 promoter region. The relationship between histone hyperacetylation and the binding of TBP was assayed in vitro using an in vivo-assembled nucleosomal array over the pS2 promoter. With chromatin in its basal state, the binding of TBP to the pS2 TATA sequence at the edge of NUC T was severely restricted, consistent with our in vivo data. Acetylation of the core histones facilitated the binding of TBP to this nucleosomal TATA sequence. Therefore, we demonstrate that one specific, functional consequence of induced histone acetylation at a native promoter is the alleviation of nucleosome-mediated repression of the binding of TBP. Our data support a fundamental role for histone acetylation at genomic promoters in transcriptional activation by nuclear receptors and provide a general mechanism for rapid and reversible transcriptional activation from a chromatin template. PMID:11158325

  2. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    PubMed Central

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-01-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks. PMID:27161996

  3. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks.

    PubMed

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-01-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks. PMID:27161996

  4. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    NASA Astrophysics Data System (ADS)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  5. Detection of an endothelin-1-binding protein complex by low temperature SDS-PAGE

    SciTech Connect

    Takasuka, T.; Horii, I.; Furuichi, Y.; Watanabe, T. )

    1991-04-15

    We found that the complex of ET-1 and its binding protein was stable enough to be separated by SDS-PAGE when electrophoresis was run at a low temperature. Cross-linking was not necessary for the detection of {sup 125}I-ET-1 and its binding protein complex by autoradiography. This simple method could be used in qualitative (estimation of apparent molecular weight of ET-1 binding protein) and quantitative (determination of relative content of ET-binding protein) analysis of the ET-binding protein complex. ET-binding protein complexes of various animal species and organs were investigated by this method.

  6. Manipulating Cofactor Binding Thermodynamics in an Artificial Oxygen Transport Protein

    PubMed Central

    Zhang, Lei; Ross Anderson, J. L.; Ahmed, Ismail; Norman, Jessica A.; Negron, Christopher; Mutter, Andrew C.; Dutton, P. Leslie; Koder, Ronald L.

    2013-01-01

    We report the mutational analysis of an artificial oxygen transport protein, HP-7, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which is uncharged, increases the affinity of the distal histidine ligand by a factor of thirteen. Paradoxically, it also decreases heme binding affinity by a factor of five in the reduced state and sixty in the oxidized state. Application of a three-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. PMID:22004125

  7. Evidence of Conformational Selection Driving the Formation of Ligand Binding Sites in Protein-Protein Interfaces

    PubMed Central

    Bohnuud, Tanggis; Kozakov, Dima; Vajda, Sandor

    2014-01-01

    Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some p