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Sample records for gp64 protein analysis

  1. Promoter analysis of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum.

    PubMed

    Takaoka, N; Fukuzawa, M; Saito, T; Sakaitani, T; Ochiai, H

    1999-10-28

    We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position -47 to -37 and to an initiator (Inr, PyPyCAPyPyPyPy) at position -3 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the beta-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between -187 and -62 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium.

  2. Autographa californica multiple nucleopolyhedrovirus GP64 protein: Analysis of domain I and V amino acid interactions and membrane fusion activity

    SciTech Connect

    Yu, Qianlong; Blissard, Gary W.; Liu, Tong-Xian; Li, Zhaofei

    2016-01-15

    The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.

  3. A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein

    PubMed Central

    Ardisson-Araújo, Daniel M. P.; Melo, Fernando L.; Clem, Rollie J.; Wolff, José L. C.

    2015-01-01

    The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage. PMID:26537678

  4. Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

    PubMed Central

    Garry, Courtney E; Garry, Robert F

    2008-01-01

    Background Members of the Baculoviridae encode two types of proteins that mediate virus:cell membrane fusion and penetration into the host cell. Alignments of primary amino acid sequences indicate that baculovirus fusion proteins of group I nucleopolyhedroviruses (NPV) form the GP64 superfamily. The structure of these viral penetrenes has not been determined. The GP64 superfamily includes the glycoprotein (GP) encoded by members of the Thogotovirus genus of the Orthomyxoviridae. The entry proteins of other baculoviruses, group II NPV and granuloviruses, are class I penetrenes. Results Class III penetrenes encoded by members of the Rhabdoviridae and Herpesviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Similar sequences and structural/functional motifs that characterize class III penetrenes are located collinearly in GP64 of group I baculoviruses and related glycoproteins encoded by thogotoviruses. Structural models based on a prototypic class III penetrene, vesicular stomatitis virus glycoprotein (VSV G), were established for Thogoto virus (THOV) GP and Autographa california multiple NPV (AcMNPV) GP64 demonstrating feasible cysteine linkages. Glycosylation sites in THOV GP and AcMNPV GP64 appear in similar model locations to the two glycosylation sites of VSV G. Conclusion These results suggest that proteins in the GP64 superfamily are class III penetrenes. PMID:18282283

  5. A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

    SciTech Connect

    Katou, Yasuhiro; Yamada, Hayato; Ikeda, Motoko; Kobayashi, Michihiro

    2010-09-01

    We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.

  6. Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein

    SciTech Connect

    Zhou Jian; Blissard, Gary W. . E-mail: gwb1@cornell.edu

    2006-09-01

    The envelope glycoprotein GP64 of Autographa californica nucleopolyhedrovirus (AcMNPV) is necessary and sufficient for the acid-induced membrane fusion activity that is required for fusion of the budded virus (BV) envelope and the endosome membrane during virus entry. Infectivity of the budded virus (BV) is neutralized by AcV1, a monoclonal antibody (MAb) directed against GP64. Prior studies indicated that AcV1 recognizes a conformational epitope and does not inhibit virus attachment to the cell, but instead inhibits entry at a step following virus attachment. We found that AcV1 recognition of GP64 was lost upon exposure of GP64 to low pH (pH 4.5) and restored by returning GP64 to pH 6.2. In addition, the AcV1 epitope was lost upon denaturation of GP64 in SDS, but the AcV1 epitope was restored by refolding the protein in the absence of SDS. Using truncated GP64 proteins expressed in insect cells, we mapped the AcV1 epitope to a 24 amino acid region in the central variable domain of GP64. When sequences within the mapped AcV1 epitope were substituted with a c-Myc epitope and the resulting construct was used to replace wt GP64 in recombinant AcMNPV viruses, the modified GP64 protein appeared to function normally. However, an anti-c-Myc monoclonal antibody did not neutralize infectivity of those viruses. Because binding of the c-Myc MAb to the same site in the GP64 sequence did not result in neutralization, these studies suggest that AcV1 neutralization may result from a specific structural constraint caused by AcV1 binding and not simply by steric hindrance caused by antibody binding at this position in GP64.

  7. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    PubMed Central

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

  8. A cholesterol recognition amino acid consensus domain in GP64 fusion protein facilitates anchoring of baculovirus to mammalian cells.

    PubMed

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R; Sampieri, Alicia; Vaca, Luis

    2013-11-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

  9. A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells.

    PubMed

    Wu, Chunxiao; Wang, Shu

    2012-01-01

    Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

  10. Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

    PubMed Central

    Oakland, Mayumi; Maury, Wendy; McCray, Paul B; Sinn, Patrick L

    2013-01-01

    Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN) responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV) in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction. PMID:23360952

  11. A MicroRNA-regulated and GP64-pseudotyped Lentiviral Vector Mediates Stable Expression of FVIII in a Murine Model of Hemophilia A

    PubMed Central

    Matsui, Hideto; Hegadorn, Carol; Ozelo, Margareth; Burnett, Erin; Tuttle, Angie; Labelle, Andrea; McCray, Paul B; Naldini, Luigi; Brown, Brian; Hough, Christine; Lillicrap, David

    2011-01-01

    The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system. PMID:21285959

  12. Functional Analysis of the Putative Fusion Domain of the Baculovirus Envelope Fusion Protein F

    PubMed Central

    Westenberg, Marcel; Veenman, Frank; Roode, Els C.; Goldbach, Rob W.; Vlak, Just M.; Zuidema, Douwe

    2004-01-01

    Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F1. The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect. PMID:15194771

  13. Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells

    PubMed Central

    Slack, J. M.; Blissard, G. W.

    2013-01-01

    Summary A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, the enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion. Spodoptera frugiperda (Sf9) insect cells expressing a chimeric Lac Repressor-IE1 protein were fused to Sf9 cells containing an EGFP reporter construct under the control of a responsive lac operator containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity. Sf9 cells expressing the Orgyia pseudotsugata Multicapsid Nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain. PMID:11562545

  14. Analysis of secreted proteins.

    PubMed

    Severino, Valeria; Farina, Annarita; Chambery, Angela

    2013-01-01

    Most biological processes including growth, proliferation, differentiation, and apoptosis are coordinated by tightly regulated signaling pathways, which also involve secreted proteins acting in an autocrine and/or paracrine manner. In addition, extracellular signaling molecules affect local niche biology and influence the cross-talking with the surrounding tissues. The understanding of this molecular language may provide an integrated and broader view of cellular regulatory networks under physiological and pathological conditions. In this context, the profiling at a global level of cell secretomes (i.e., the subpopulations of a proteome secreted from the cell) has become an active area of research. The current interest in secretome research also deals with its high potential for the biomarker discovery and the identification of new targets for therapeutic strategies. Several proteomic and mass spectrometry platforms and methodologies have been applied to secretome profiling of conditioned media of cultured cell lines and primary cells. Nevertheless, the analysis of secreted proteins is still a very challenging task, because of the technical difficulties that may hamper the subsequent mass spectrometry analysis. This chapter describes a typical workflow for the analysis of proteins secreted by cultured cells. Crucial issues related to cell culture conditions for the collection of conditioned media, secretome preparation, and mass spectrometry analysis are discussed. Furthermore, an overview of quantitative LC-MS-based approaches, computational tools for data analysis, and strategies for validation of potential secretome biomarkers is also presented.

  15. Statistical Analysis of Protein Ensembles

    NASA Astrophysics Data System (ADS)

    Máté, Gabriell; Heermann, Dieter

    2014-04-01

    As 3D protein-configuration data is piling up, there is an ever-increasing need for well-defined, mathematically rigorous analysis approaches, especially that the vast majority of the currently available methods rely heavily on heuristics. We propose an analysis framework which stems from topology, the field of mathematics which studies properties preserved under continuous deformations. First, we calculate a barcode representation of the molecules employing computational topology algorithms. Bars in this barcode represent different topological features. Molecules are compared through their barcodes by statistically determining the difference in the set of their topological features. As a proof-of-principle application, we analyze a dataset compiled of ensembles of different proteins, obtained from the Ensemble Protein Database. We demonstrate that our approach correctly detects the different protein groupings.

  16. Wavelet Analysis of Protein Motion

    PubMed Central

    BENSON, NOAH C.

    2014-01-01

    As high-throughput molecular dynamics simulations of proteins become more common and the databases housing the results become larger and more prevalent, more sophisticated methods to quickly and accurately mine large numbers of trajectories for relevant information will have to be developed. One such method, which is only recently gaining popularity in molecular biology, is the continuous wavelet transform, which is especially well-suited for time course data such as molecular dynamics simulations. We describe techniques for the calculation and analysis of wavelet transforms of molecular dynamics trajectories in detail and present examples of how these techniques can be useful in data mining. We demonstrate that wavelets are sensitive to structural rearrangements in proteins and that they can be used to quickly detect physically relevant events. Finally, as an example of the use of this approach, we show how wavelet data mining has led to a novel hypothesis related to the mechanism of the protein γδ resolvase. PMID:25484480

  17. Quantitative analysis of glycated proteins.

    PubMed

    Priego-Capote, Feliciano; Ramírez-Boo, María; Finamore, Francesco; Gluck, Florent; Sanchez, Jean-Charles

    2014-02-07

    The proposed protocol presents a comprehensive approach for large-scale qualitative and quantitative analysis of glycated proteins (GP) in complex biological samples including biological fluids and cell lysates such as plasma and red blood cells. The method, named glycation isotopic labeling (GIL), is based on the differential labeling of proteins with isotopic [(13)C6]-glucose, which supports quantitation of the resulting glycated peptides after enzymatic digestion with endoproteinase Glu-C. The key principle of the GIL approach is the detection of doublet signals for each glycated peptide in MS precursor scanning (glycated peptide with in vivo [(12)C6]- and in vitro [(13)C6]-glucose). The mass shift of the doublet signals is +6, +3 or +2 Da depending on the peptide charge state and the number of glycation sites. The intensity ratio between doublet signals generates quantitative information of glycated proteins that can be related to the glycemic state of the studied samples. Tandem mass spectrometry with high-energy collisional dissociation (HCD-MS2) and data-dependent methods with collision-induced dissociation (CID-MS3 neutral loss scan) are used for qualitative analysis.

  18. Analysis of oxidative modification of proteins.

    PubMed

    Yan, Liang-Jun

    2009-02-01

    Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [(14)C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-L-isoaspartyl methyltransferase that converts aberrant L-isoaspartyl residues in peptides and proteins to normal aspartyl residues.

  19. Analysis of oxidative modification of proteins.

    PubMed

    Yan, Liang-Jun

    2009-04-01

    Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [(14)C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-L-isoaspartyl methyltransferase that converts aberrant L-isoaspartyl residues in peptides and proteins to normal aspartyl residues.

  20. Protein-protein interactions: methods for detection and analysis.

    PubMed Central

    Phizicky, E M; Fields, S

    1995-01-01

    The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques. PMID:7708014

  1. Proteomic analysis of integral plasma membrane proteins.

    PubMed

    Zhao, Yingxin; Zhang, Wei; Kho, Yoonjung; Zhao, Yingming

    2004-04-01

    Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

  2. Protein-protein interaction network analysis of cirrhosis liver disease.

    PubMed

    Safaei, Akram; Rezaei Tavirani, Mostafa; Arefi Oskouei, Afsaneh; Zamanian Azodi, Mona; Mohebbi, Seyed Reza; Nikzamir, Abdol Rahim

    2016-01-01

    Evaluation of biological characteristics of 13 identified proteins of patients with cirrhotic liver disease is the main aim of this research. In clinical usage, liver biopsy remains the gold standard for diagnosis of hepatic fibrosis. Evaluation and confirmation of liver fibrosis stages and severity of chronic diseases require a precise and noninvasive biomarkers. Since the early detection of cirrhosis is a clinical problem, achieving a sensitive, specific and predictive novel method based on biomarkers is an important task. Essential analysis, such as gene ontology (GO) enrichment and protein-protein interactions (PPI) was undergone EXPASy, STRING Database and DAVID Bioinformatics Resources query. Based on GO analysis, most of proteins are located in the endoplasmic reticulum lumen, intracellular organelle lumen, membrane-enclosed lumen, and extracellular region. The relevant molecular functions are actin binding, metal ion binding, cation binding and ion binding. Cell adhesion, biological adhesion, cellular amino acid derivative, metabolic process and homeostatic process are the related processes. Protein-protein interaction network analysis introduced five proteins (fibroblast growth factor receptor 4, tropomyosin 4, tropomyosin 2 (beta), lectin, Lectin galactoside-binding soluble 3 binding protein and apolipoprotein A-I) as hub and bottleneck proteins. Our result indicates that regulation of lipid metabolism and cell survival are important biological processes involved in cirrhosis disease. More investigation of above mentioned proteins will provide a better understanding of cirrhosis disease.

  3. Protein-protein interaction network analysis of cirrhosis liver disease

    PubMed Central

    Safaei, Akram; Rezaei Tavirani, Mostafa; Arefi Oskouei, Afsaneh; Zamanian Azodi, Mona; Mohebbi, Seyed Reza; Nikzamir, Abdol Rahim

    2016-01-01

    Aim: Evaluation of biological characteristics of 13 identified proteins of patients with cirrhotic liver disease is the main aim of this research. Background: In clinical usage, liver biopsy remains the gold standard for diagnosis of hepatic fibrosis. Evaluation and confirmation of liver fibrosis stages and severity of chronic diseases require a precise and noninvasive biomarkers. Since the early detection of cirrhosis is a clinical problem, achieving a sensitive, specific and predictive novel method based on biomarkers is an important task. Methods: Essential analysis, such as gene ontology (GO) enrichment and protein-protein interactions (PPI) was undergone EXPASy, STRING Database and DAVID Bioinformatics Resources query. Results: Based on GO analysis, most of proteins are located in the endoplasmic reticulum lumen, intracellular organelle lumen, membrane-enclosed lumen, and extracellular region. The relevant molecular functions are actin binding, metal ion binding, cation binding and ion binding. Cell adhesion, biological adhesion, cellular amino acid derivative, metabolic process and homeostatic process are the related processes. Protein-protein interaction network analysis introduced five proteins (fibroblast growth factor receptor 4, tropomyosin 4, tropomyosin 2 (beta), lectin, Lectin galactoside-binding soluble 3 binding protein and apolipoprotein A-I) as hub and bottleneck proteins. Conclusion: Our result indicates that regulation of lipid metabolism and cell survival are important biological processes involved in cirrhosis disease. More investigation of above mentioned proteins will provide a better understanding of cirrhosis disease. PMID:27099671

  4. Protein intrinsic disorder toolbox for comparative analysis of viral proteins

    PubMed Central

    Goh, Gerard Kian-Meng; Dunker, A Keith; Uversky, Vladimir N

    2008-01-01

    To examine the usefulness of protein disorder predictions as a tool for the comparative analysis of viral proteins, a relational database has been constructed. The database includes proteins from influenza A and HIV-related viruses. Annotations include viral protein sequence, disorder prediction, structure, and function. Location of each protein within a virion, if known, is also denoted. Our analysis reveals a clear relationship between proximity to the RNA core and the percentage of predicted disordered residues for a set of influenza A virus proteins. Neuraminidases (NA) and hemagglutinin (HA) of major influenza A pandemics tend to pair in such a way that both proteins tend to be either ordered-ordered or disordered-disordered by prediction. This may be the result of these proteins evolving from being lipid-associated. High abundance of intrinsic disorder in envelope and matrix proteins from HIV-related viruses likely represents a mechanism where HIV virions can escape immune response despite the availability of antibodies for the HIV-related proteins. This exercise provides an example showing how the combined use of intrinsic disorder predictions and relational databases provides an improved understanding of the functional and structural behaviour of viral proteins. PMID:18831795

  5. A protein structure data and analysis system.

    PubMed

    Tian, Hao; Sunderraman, Rajshekhar; Weber, Irene; Wang, Haibin; Yang, Hong

    2005-01-01

    In this paper, we present the design and implementation of a protein structure data and analysis system that is only used in the lab for analyzing the proprietary data. It is capable of storing public protein data, such as the data in Protein Data Bank (PDB) [1], and life scientists' proprietary data. This toolkit is targeted at life scientists who want to maintain proprietary protein structure data (may be incomplete), to search and query publicly known protein structures and to compare their structure data with others. The comparison functions can be used to find structure differences between two proteins at atom level, especially in mutant versions of proteins. The system can also be used as a tool of choosing better protein structure template in new protein's tertiary structure prediction. The system is developed in Java and the protein data is stored in a relational database (Oracle 9i).

  6. NAPS: Network Analysis of Protein Structures

    PubMed Central

    Chakrabarty, Broto; Parekh, Nita

    2016-01-01

    Traditionally, protein structures have been analysed by the secondary structure architecture and fold arrangement. An alternative approach that has shown promise is modelling proteins as a network of non-covalent interactions between amino acid residues. The network representation of proteins provide a systems approach to topological analysis of complex three-dimensional structures irrespective of secondary structure and fold type and provide insights into structure-function relationship. We have developed a web server for network based analysis of protein structures, NAPS, that facilitates quantitative and qualitative (visual) analysis of residue–residue interactions in: single chains, protein complex, modelled protein structures and trajectories (e.g. from molecular dynamics simulations). The user can specify atom type for network construction, distance range (in Å) and minimal amino acid separation along the sequence. NAPS provides users selection of node(s) and its neighbourhood based on centrality measures, physicochemical properties of amino acids or cluster of well-connected residues (k-cliques) for further analysis. Visual analysis of interacting domains and protein chains, and shortest path lengths between pair of residues are additional features that aid in functional analysis. NAPS support various analyses and visualization views for identifying functional residues, provide insight into mechanisms of protein folding, domain-domain and protein–protein interactions for understanding communication within and between proteins. URL:http://bioinf.iiit.ac.in/NAPS/. PMID:27151201

  7. Stochastic model for protein flexibility analysis.

    PubMed

    Xia, Kelin; Wei, Guo-Wei

    2013-12-01

    Protein flexibility is an intrinsic property and plays a fundamental role in protein functions. Computational analysis of protein flexibility is crucial to protein function prediction, macromolecular flexible docking, and rational drug design. Most current approaches for protein flexibility analysis are based on Hamiltonian mechanics. We introduce a stochastic model to study protein flexibility. The essential idea is to analyze the free induction decay of a perturbed protein structural probability, which satisfies the master equation. The transition probability matrix is constructed by using probability density estimators including monotonically decreasing radial basis functions. We show that the proposed stochastic model gives rise to some of the best predictions of Debye-Waller factors or B factors for three sets of protein data introduced in the literature.

  8. A Micro-Method of Protein Analysis

    DTIC Science & Technology

    The determination of protein by means of Weichselbaum’s (1) biuret method is too inexact when dealing with small quantities of protein (less than 200...microgram/ml initial reactant), owing to the low sensitivity of the color reaction . Although we have used this method for protein analysis of...have searched for a more sensitive colorimetric method. Nielsen (3) recently reported on a method in which the Cu bound by protein in the biuret

  9. Protein Analysis with Human Hair

    SciTech Connect

    Hart, Brad; Anex, Deon; Parker, Glendon

    2016-09-07

    In an important breakthrough for the forensic science community, researchers have developed the first-ever biological identification method that exploits the information encoded in proteins of human hair.

  10. Protein Analysis with Human Hair

    ScienceCinema

    Hart, Brad; Anex, Deon; Parker, Glendon

    2016-09-09

    In an important breakthrough for the forensic science community, researchers have developed the first-ever biological identification method that exploits the information encoded in proteins of human hair.

  11. Global Proteomics Analysis of Protein Lysine Methylation.

    PubMed

    Cao, Xing-Jun; Garcia, Benjamin A

    2016-11-01

    Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non-histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites. However, recently, there has been a series of publications to perform large-scale analysis of protein lysine methylation. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  12. Microfluidic Approaches for Protein Crystal Structure Analysis.

    PubMed

    Maeki, Masatoshi; Yamaguchi, Hiroshi; Tokeshi, Manabu; Miyazaki, Masaya

    2016-01-01

    This review summarizes two microfluidic-based protein crystallization methods, protein crystallization behavior in the microfluidic devices, and their applications for X-ray crystal structure analysis. Microfluidic devices provide many advantages for protein crystallography; they require small sample volumes, provide high-throughput screening, and allow control of the protein crystallization. A droplet-based protein crystallization method is a useful technique for high-throughput screening and the formation of a single crystal without any complicated device fabrication process. Well-based microfluidic platforms also enable effective protein crystallization. This review also summarizes the protein crystal growth behavior in microfluidic devices as, is known from viewpoints of theoretical and experimental approaches. Finally, we introduce applications of microfluidic devices for on-chip crystal structure analysis.

  13. [Methods for analysis of protein-protein and protein-ligand interactions].

    PubMed

    Durech, M; Trčka, F; Vojtěšek, B; Müller, P

    2014-01-01

    In order to maintain cellular homeostasis, cellular proteins coexist in complex and variable molecular assemblies. Therefore, understanding of major physiological processes at molecular level is based on analysis of protein-protein interaction networks. Firstly, composition of the molecular assembly has to be qualitatively analyzed. In the next step, quantitative bio-chemical properties of the identified protein-protein interactions are determined. Detailed information about the protein-protein interaction interface can be obtained by crystallographic methods. Accordingly, the insight into the molecular architecture of these protein-protein complexes allows us to rationally design new synthetic compounds that specifically influence various physiological or pathological processes by targeted modulation of protein interactions. This review is focused on description of the most used methods applied in both qualitative and quantitative analysis of protein-protein interactions. Co- immunoprecipitation and affinity co- precipitation are basic methods designed for qualitative analysis of protein binding partners. Further bio-chemical analysis of the interaction requires definition of kinetic and thermodynamic parameters. Surface plasmon resonance (SPR) is used for description of affinity and kinetic profile of the interaction, fluorescence polarization (FP) method for fast determination of inhibition potential of inhibitors and isothermal titration calorimetry (ITC) for definition of thermodynamic parameters of the interaction (G, H and S). Besides the importance of uncovering the molecular basis of protein interactions for basic research, the same methodological approaches open new possibilities in rational design of novel therapeutic agents.

  14. Data Analysis Strategies for Protein Modification Identification.

    PubMed

    Fu, Yan

    2016-01-01

    Mass spectrometry-based proteomics provides a powerful tool for large-scale analysis of protein modifications. Statistical and computational analysis of mass spectrometry data is a key step in protein modification identification. This chapter presents common and advanced data analysis strategies for modification identification, including variable modification search, unrestrictive approaches for modification discovery, false discovery rate estimation and control methods, and tools for modification site localization.

  15. Global Proteomics Analysis of Protein Lysine Methylation

    PubMed Central

    Cao, Xing-Jun; Garcia, Benjamin A.

    2017-01-01

    Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins play substantial roles in a variety of functions in cells, and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs lead to tumorigenesis via their non-histone substrates. However, more studies on other PKMTs have made slow progress owing to the lack of the approaches for extensive screening of lysine methylation sites. Recently a series of publications to perform large-scale analysis of protein lysine methylation have emerged. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry. PMID:27801517

  16. Protein sectors: statistical coupling analysis versus conservation.

    PubMed

    Teşileanu, Tiberiu; Colwell, Lucy J; Leibler, Stanislas

    2015-02-01

    Statistical coupling analysis (SCA) is a method for analyzing multiple sequence alignments that was used to identify groups of coevolving residues termed "sectors". The method applies spectral analysis to a matrix obtained by combining correlation information with sequence conservation. It has been asserted that the protein sectors identified by SCA are functionally significant, with different sectors controlling different biochemical properties of the protein. Here we reconsider the available experimental data and note that it involves almost exclusively proteins with a single sector. We show that in this case sequence conservation is the dominating factor in SCA, and can alone be used to make statistically equivalent functional predictions. Therefore, we suggest shifting the experimental focus to proteins for which SCA identifies several sectors. Correlations in protein alignments, which have been shown to be informative in a number of independent studies, would then be less dominated by sequence conservation.

  17. Quantitative analysis of protein turnover in plants.

    PubMed

    Nelson, Clark J; Li, Lei; Millar, A Harvey

    2014-03-01

    Proteins are constantly being synthesised and degraded as plant cells age and as plants grow, develop and adapt the proteome. Given that plants develop through a series of events from germination to fruiting and even undertake whole organ senescence, an understanding of protein turnover as a fundamental part of this process in plants is essential. Both synthesis and degradation processes are spatially separated in a cell across its compartmented structure. The majority of protein synthesis occurs in the cytosol, while synthesis of specific components occurs inside plastids and mitochondria. Degradation of proteins occurs in both the cytosol, through the action of the plant proteasome, and in organelles and lytic structures through different protease classes. Tracking the specific synthesis and degradation rate of individual proteins can be undertaken using stable isotope feeding and the ability of peptide MS to track labelled peptide fractions over time. Mathematical modelling can be used to follow the isotope signature of newly synthesised protein as it accumulates and natural abundance proteins as they are lost through degradation. Different technical and biological constraints govern the potential for the use of (13)C, (15)N, (2)H and (18)O for these experiments in complete labelling and partial labelling strategies. Future development of quantitative protein turnover analysis will involve analysis of protein populations in complexes and subcellular compartments, assessing the effect of PTMs and integrating turnover studies into wider system biology study of plants. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Terahertz Spectroscopic Analysis of Peptides and Proteins

    NASA Astrophysics Data System (ADS)

    Falconer, Robert J.; Markelz, Andrea G.

    2012-10-01

    Spectroscopic analysis using the Terahertz frequencies between 0.1-15 THz (3-500 cm-1) has been underutilised by the biochemistry community but is starting to yield some scientifically interesting information. Analysis of structures from simple molecules like N-methylacetamide, to polyamides, peptides and relatively complex proteins provides different types of information dependant on the molecular size. The absorbance spectrum of small molecules is dominated by individual modes and specific hydrogen bonds, peptide spectra have peaks associated with secondary structure, while protein spectra are dominated by ensembles of hydrogen bonds and/or collective modes. Protein dynamics has been studied using Terahertz spectroscopy using proteins like bacteriorhodopsin, illustrating a potential application where this approach can provide complementary global dynamics information to the current nuclear magnetic resonance and fluorescence-based techniques. Analysis of higher-order protein structures like polyomavirus virus-like particles generate quite different spectra compared to their constituent parts. The presence of an extended hydration layer around proteins, first postulated to explain data generated using p-germanium spectroscopy may present a particularly interesting opportunity to better understand protein's complex interaction with water and small solutes in an aqueous environment. The practical aspects of Terahertz spectroscopy including sample handling, the use of molecular dynamics simulation and orthogonal experiment design are also discussed.

  19. Automated Protein Assay Using Flow Injection Analysis

    NASA Astrophysics Data System (ADS)

    Wolfe, Carrie A. C.; Oates, Matthew R.; Hage, David S.

    1998-08-01

    The technique of flow injection analysis (FIA) is a common instrumental method used in detecting a variety of chemical and biological agents. This paper describes an undergraduate laboratory that uses FIA to perform a bicinchoninic acid (BCA) colorimetric assay for quantitating protein samples. The method requires less than 2 min per sample injection and gives a response over a broad range of protein concentrations. This method can be used in instrumental analysis labs to illustrate the principles and use of FIA, or as a means for introducing students to common methods employed in the analysis of biological agents.

  20. Blood proteins analysis by Raman spectroscopy method

    NASA Astrophysics Data System (ADS)

    Artemyev, D. N.; Bratchenko, I. A.; Khristoforova, Yu. A.; Lykina, A. A.; Myakinin, O. O.; Kuzmina, T. P.; Davydkin, I. L.; Zakharov, V. P.

    2016-04-01

    This work is devoted to study the possibility of plasma proteins (albumin, globulins) concentration measurement using Raman spectroscopy setup. The blood plasma and whole blood were studied in this research. The obtained Raman spectra showed significant variation of intensities of certain spectral bands 940, 1005, 1330, 1450 and 1650 cm-1 for different protein fractions. Partial least squares regression analysis was used for determination of correlation coefficients. We have shown that the proposed method represents the structure and biochemical composition of major blood proteins.

  1. Analysis of protein aggregation kinetics.

    PubMed

    Ferrone, F

    1999-01-01

    Given a set of kinetic data, then, the preceding discussions suggest the following approach to its analysis. 1. For purposes of establishing the reaction, ignore the final stages and concentrate on the initial 10-20% of the reaction at first. A globally optimized model may be based on a faulty assumption for the initial steps. Thus, although the whole data set may look reasonably well fit, the reaction could be misrepresented, and thus the fit unhelpful if accuracy at the later stages has come at the expense of the initial phase of the reaction. 2. What is the time course of the initial reaction? (A) Is the reaction exponential? Exponential growth gives dramatic lag times (see Fig. 3), whereas nonexponential "lag times" have a visible signal from time 0 (i.e., Fig. 2). If the data set shows the abrupt appearance of signals after a period of quiescence, the chances are excellent that the time course is exponential. High sensitivity measurement of the signal at times during the lag phase should be used to confirm the exponential nature quantitatively. Exponential reactions mean a secondary pathway is operative. (a) A cascade (tn) can look similar to an exponential, but may proceed from a multistep single-path reaction. Thus the exponential needs to be ascertained with some accuracy. (b) It is possible that some or all of the lag results from a stochastic process, i.e., formation of a single nucleus being observed. This, however, is likely to be accompanied by a secondary process, as few techniques are sensitive enough to detect a single polymer at a time, and having one nucleus form many polymers is a hallmark of a secondary process. Thus, the reproducibility of the kinetics must be established to rule out stochastics. If data show wide variation, stochastic methods as described earlier may be employed. (c) Given a secondary process, one must separate the primary nucleation process from the secondary process (by stochastic means or by use of the product B2A, as

  2. Predictive and comparative analysis of Ebolavirus proteins

    PubMed Central

    Cong, Qian; Pei, Jimin; Grishin, Nick V

    2015-01-01

    Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus. PMID:26158395

  3. Spectral Analysis of a Protein Conformational Switch

    NASA Astrophysics Data System (ADS)

    Rackovsky, S.

    2011-06-01

    The existence of conformational switching in proteins, induced by single amino acid mutations, presents an important challenge to our understanding of the physics of protein folding. Sequence-local methods, commonly used to detect structural homology, are incapable of accounting for this phenomenon. We examine a set of proteins, derived from the GA and GB domains of Streptococcus protein G, which are known to show a dramatic conformational change as a result of single-residue replacement. It is shown that these sequences, which are almost identical locally, can have very different global patterns of physical properties. These differences are consistent with the observed complete change in conformation. These results suggest that sequence-local methods for identifying structural homology can be misleading. They point to the importance of global sequence analysis in understanding sequence-structure relationships.

  4. Complementary Proteomic Analysis of Protein Complexes

    PubMed Central

    Greco, Todd M.; Miteva, Yana; Conlon, Frank L.; Cristea, Ileana M.

    2013-01-01

    Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we describe a complementary proteomic approach optimized for immunoisolated protein complexes. As the relative complexity, abundance, and physiochemical properties of proteins can vary significantly between samples, we have provided (1) complementary sample preparation workflows, (2) detailed steps for HPLC and mass spectrometric method development, and (3) a bioinformatic workflow that provides confident peptide/protein identification paired with unbiased functional gene ontology analysis. This protocol can also be extended for characterization of larger complexity samples from whole cell or tissue Xenopus proteomes. PMID:22956100

  5. Nanobiocatalysis for protein digestion in proteomic analysis

    SciTech Connect

    Kim, Jungbae; Kim, Byoung Chan; Lopez-Ferrer, Daniel; Petritis, Konstantinos; Smith, Richard D.

    2010-02-01

    The process of protein digestion is a critical step for successful protein identification in the bottom-up proteomic analysis. To substitute the present practice of in-solution protein digestion, which is long, tedious, and difficult to automate, a lot of efforts have been dedicated for the development of a rapid, recyclable and automated digestion system. Recent advances of nanobiocatalytic approaches have improved the performance of protein digestion by using various nanomaterials such as nanoporous materials, magnetic nanoparticles, and polymer nanofibers. Especially, the unprecedented success of trypsin stabilization in the form of trypsin-coated nanofibers, showing no activity decrease under repeated uses for one year and retaining good resistance to proteolysis, has demonstrated its great potential to be employed in the development of automated, high-throughput, and on-line digestion systems. This review discusses recent developments of nanobiocatalytic approaches for the improved performance of protein digestion in speed, detection sensitivity, recyclability, and trypsin stability. In addition, we also introduce the protein digestions under unconventional energy inputs for protein denaturation and the development of microfluidic enzyme reactors that can benefit from recent successes of these nanobiocatalytic approaches.

  6. Protein Cross-Linking Capillary Electrophoresis for Protein-Protein Interaction Analysis.

    PubMed

    Ouimet, Claire M; Shao, Hao; Rauch, Jennifer N; Dawod, Mohamed; Nordhues, Bryce; Dickey, Chad A; Gestwicki, Jason E; Kennedy, Robert T

    2016-08-16

    Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs.

  7. Comparative analysis of five protein-protein interaction corpora.

    PubMed

    Pyysalo, Sampo; Airola, Antti; Heimonen, Juho; Björne, Jari; Ginter, Filip; Salakoski, Tapio

    2008-04-11

    Growing interest in the application of natural language processing methods to biomedical text has led to an increasing number of corpora and methods targeting protein-protein interaction (PPI) extraction. However, there is no general consensus regarding PPI annotation and consequently resources are largely incompatible and methods are difficult to evaluate. We present the first comparative evaluation of the diverse PPI corpora, performing quantitative evaluation using two separate information extraction methods as well as detailed statistical and qualitative analyses of their properties. For the evaluation, we unify the corpus PPI annotations to a shared level of information, consisting of undirected, untyped binary interactions of non-static types with no identification of the words specifying the interaction, no negations, and no interaction certainty. We find that the F-score performance of a state-of-the-art PPI extraction method varies on average 19 percentage units and in some cases over 30 percentage units between the different evaluated corpora. The differences stemming from the choice of corpus can thus be substantially larger than differences between the performance of PPI extraction methods, which suggests definite limits on the ability to compare methods evaluated on different resources. We analyse a number of potential sources for these differences and identify factors explaining approximately half of the variance. We further suggest ways in which the difficulty of the PPI extraction tasks codified by different corpora can be determined to advance comparability. Our analysis also identifies points of agreement and disagreement in PPI corpus annotation that are rarely explicitly stated by the authors of the corpora. Our comparative analysis uncovers key similarities and differences between the diverse PPI corpora, thus taking an important step towards standardization. In the course of this study we have created a major practical contribution in

  8. Statistical analysis and prediction of protein-protein interfaces.

    PubMed

    Bordner, Andrew J; Abagyan, Ruben

    2005-08-15

    Predicting protein-protein interfaces from a three-dimensional structure is a key task of computational structural proteomics. In contrast to geometrically distinct small molecule binding sites, protein-protein interface are notoriously difficult to predict. We generated a large nonredundant data set of 1494 true protein-protein interfaces using biological symmetry annotation where necessary. The data set was carefully analyzed and a Support Vector Machine was trained on a combination of a new robust evolutionary conservation signal with the local surface properties to predict protein-protein interfaces. Fivefold cross validation verifies the high sensitivity and selectivity of the model. As much as 97% of the predicted patches had an overlap with the true interface patch while only 22% of the surface residues were included in an average predicted patch. The model allowed the identification of potential new interfaces and the correction of mislabeled oligomeric states.

  9. Sequence analysis of the complete genome of Trichoplusia ni single nucleopolyhedrovirus and the identification of a baculoviral photolyase gene

    SciTech Connect

    Willis, Leslie G.; Siepp, Robyn; Stewart, Taryn M.; Erlandson, Martin A.; Theilmann, David A. . E-mail: TheilmannD@agr.gc.ca

    2005-08-01

    The genome of the Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), a group II NPV which infects the cabbage looper (T. ni), has been completely sequenced and analyzed. The TnSNPV DNA genome consists of 134,394 bp and has an overall G + C content of 39%. Gene analysis predicted 144 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. Comparisons with previously sequenced baculoviruses indicate that 119 TnSNPV ORFs were homologues of previously reported viral gene sequences. Ninety-four TnSNPV ORFs returned an Autographa californica multiple NPV (AcMNPV) homologue while 25 ORFs returned poor or no sequence matches with the current databases. A putative photolyase gene was also identified that had highest amino acid identity to the photolyase genes of Chrysodeixis chalcites NPV (ChchNPV) (47%) and Danio rerio (zebrafish) (40%). In addition unlike all other baculoviruses no obvious homologous repeat (hr) sequences were identified. Comparison of the TnSNPV and AcMNPV genomes provides a unique opportunity to examine two baculoviruses that are highly virulent for a common insect host (T. ni) yet belong to diverse baculovirus taxonomic groups and possess distinct biological features. In vitro fusion assays demonstrated that the TnSNPV F protein induces membrane fusion and syncytia formation and were compared to syncytia formed by AcMNPV GP64.

  10. Mutant analysis, protein-protein interactions and subcellular localization of the Arabidopsis B sister (ABS) protein.

    PubMed

    Kaufmann, Kerstin; Anfang, Nicole; Saedler, Heinz; Theissen, Günter

    2005-09-01

    Recently, close relatives of class B floral homeotic genes, termed B(sister) genes, have been identified in both angiosperms and gymnosperms. In contrast to the B genes themselves, B(sister) genes are exclusively expressed in female reproductive organs, especially in the envelopes or integuments surrounding the ovules. This suggests an important ancient function in ovule or seed development for B(sister) genes, which has been conserved for about 300 million years. However, investigation of the first loss-of-function mutant for a B(sister) gene (ABS/TT16 from Arabidopsis) revealed only a weak phenotype affecting endothelium formation. Here, we present an analysis of two additional mutant alleles, which corroborates this weak phenotype. Transgenic plants that ectopically express ABS show changes in the growth and identity of floral organs, suggesting that ABS can interact with floral homeotic proteins. Yeast-two-hybrid and three-hybrid analyses indicated that ABS can form dimers with SEPALLATA (SEP) floral homeotic proteins and multimeric complexes that also include the AGAMOUS-like proteins SEEDSTICK (STK) or SHATTERPROOF1/2 (SHP1, SHP2). These data suggest that the formation of multimeric transcription factor complexes might be a general phenomenon among MIKC-type MADS-domain proteins in angiosperms. Heterodimerization of ABS with SEP3 was confirmed by gel retardation assays. Fusion proteins tagged with CFP (Cyan Fluorescent Protein) and YFP (Yellow Fluorescent Protein) in Arabidopsis protoplasts showed that ABS is localized in the nucleus. Phylogenetic analysis revealed the presence of a structurally deviant, but closely related, paralogue of ABS in the Arabidopsis genome. Thus the evolutionary developmental genetics of B(sister) genes can probably only be understood as part of a complex and redundant gene network that may govern ovule formation in a conserved manner, which has yet to be fully explored.

  11. FT-IR analysis of phosphorylated protein

    NASA Astrophysics Data System (ADS)

    Ishii, Katsunori; Yoshihashi, Sachiko S.; Chihara, Kunihiro; Awazu, Kunio

    2004-09-01

    Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070cm-1 (9.4μm) with Fourier Transform Infrared Spectrometer (FT-IR). This study indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm-1 with FT-IR method. As long as target peptides have the same amino acid sequence, it is possible to identify the phosphorylated sites (threonine, serine and tyrosine).

  12. Epock: rapid analysis of protein pocket dynamics.

    PubMed

    Laurent, Benoist; Chavent, Matthieu; Cragnolini, Tristan; Dahl, Anna Caroline E; Pasquali, Samuela; Derreumaux, Philippe; Sansom, Mark S P; Baaden, Marc

    2015-05-01

    The volume of an internal protein pocket is fundamental to ligand accessibility. Few programs that compute such volumes manage dynamic data from molecular dynamics (MD) simulations. Limited performance often prohibits analysis of large datasets. We present Epock, an efficient command-line tool that calculates pocket volumes from MD trajectories. A plugin for the VMD program provides a graphical user interface to facilitate input creation, run Epock and analyse the results. Epock C++ source code, Python analysis scripts, VMD Tcl plugin, documentation and installation instructions are freely available at http://epock.bitbucket.org. benoist.laurent@gmail.com or baaden@smplinux.de Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

  13. Baculovirus surface display of sigmaC and sigmaB proteins of avian reovirus and immunogenicity of the displayed proteins in a mouse model.

    PubMed

    Lin, Yueh H; Lee, Long H; Shih, Wen L; Hu, Yu C; Liu, Hung J

    2008-11-25

    Avian reovirus (ARV), an important pathogen in poultry, causes arthritis, chronic respiratory disease, and malabsorption syndrome that cause considerable economic losses to the poultry industry. In present study, we have succeeded in construction of a universal baculovirus surface display system (UBSDS) that can display different foreign proteins on the envelope of baculovirus. Sequences encoding the signal peptide (SS), transmembrane domain (TM), and cytoplasmic domain (CTD) derived from the gp64 protein of baculovirus and histidine tag, respectively were inserted into the pBacCE vector. Four restriction enzyme sites between the histidine tag and gp64 transmembrane domain were established for expression of different foreign proteins. The transmembrane domain and CTD of gp64 in the platform were designed in order to improve stability and quantity of foreign proteins on the envelope of baculovirus. The sigmaC and sigmaB proteins of ARV are known to elicit neutralizing antibodies against ARV. The UBSDS was therefore used to express sigmaC and sigmaB proteins on the envelope of baculovirus. Two recombinant baculoviruses BacSC-sigmaC and BacSC-sigmaB have been successfully constructed. After infection, both His6-tagged recombinant sigmaC (rsigmaC) and sigmaB (rsigmaB) proteins were displayed on the envelope of recombinant baculoviruses and the recombinant viral proteins were anchored on the plasma membrane of Sf-9 cells, as revealed by immunofluorescence staining (IFS) and confocal microscopy. The antigenicity of rsigmaC and rsigmaB proteins was demonstrated by Western blotting assay. Immunogold electron microscopy demonstrated that both recombinant viruses displayed rsigmaC and rsigmaB proteins on the viral surface. Immunization of BALB/c mice with recombinant viruses, demonstrated that serum from the BacSC-sigmaC and BacSC-sigmaB treated models had significant higher levels of virus neutralization activities than the control groups. This demonstrates that the

  14. Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.

    PubMed

    Sinn, Patrick L; Burnight, Erin R; Hickey, Melissa A; Blissard, Gary W; McCray, Paul B

    2005-10-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

  15. Mid-infrared spectroscopy for protein analysis: potential and challenges.

    PubMed

    López-Lorente, Ángela I; Mizaikoff, Boris

    2016-04-01

    Mid-infrared (MIR) spectroscopy investigates the interaction of MIR photons with both organic and inorganic molecules via the excitation of vibrational and rotational modes, providing inherent molecular selectivity. In general, infrared (IR) spectroscopy is particularly sensitive to protein structure and structural changes via vibrational resonances originating from the polypeptide backbone or side chains; hence information on the secondary structure of proteins can be obtained in a label-free fashion. In this review, the challenges for IR spectroscopy for protein analysis are discussed as are the potential and limitations of different IR spectroscopic techniques enabling protein analysis. In particular, the amide I spectral range has been widely used to study protein secondary structure, conformational changes, protein aggregation, protein adsorption, and the formation of amyloid fibrils. In addition to representative examples of the potential of IR spectroscopy in various fields related to protein analysis, the potential of protein analysis taking advantage of miniaturized MIR systems, including waveguide-enhanced MIR sensors, is detailed.

  16. Assigning protein functions by comparative genome analysis protein phylogenetic profiles

    DOEpatents

    Pellegrini, Matteo; Marcotte, Edward M.; Thompson, Michael J.; Eisenberg, David; Grothe, Robert; Yeates, Todd O.

    2003-05-13

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  17. Microcanonical versus Canonical Analysis of Protein Folding

    NASA Astrophysics Data System (ADS)

    Hernández-Rojas, J.; Gomez Llorente, J. M.

    2008-06-01

    The microcanonical analysis is shown to be a powerful tool to characterize the protein folding transition and to neatly distinguish between good and bad folders. An off-lattice model with parameter chosen to represent polymers of these two types is used to illustrate this approach. Both canonical and microcanonical ensembles are employed. The required calculations were performed using parallel tempering Monte Carlo simulations. The most revealing features of the folding transition are related to its first-order-like character, namely, the S-bend pattern in the caloric curve, which gives rise to negative microcanonical specific heats, and the bimodality of the energy distribution function at the transition temperatures. Models for a good folder are shown to be quite robust against perturbations in the interaction potential parameters.

  18. Stability analysis of an autocatalytic protein model

    NASA Astrophysics Data System (ADS)

    Lee, Julian

    2016-05-01

    A self-regulatory genetic circuit, where a protein acts as a positive regulator of its own production, is known to be the simplest biological network with a positive feedback loop. Although at least three components—DNA, RNA, and the protein—are required to form such a circuit, stability analysis of the fixed points of this self-regulatory circuit has been performed only after reducing the system to a two-component system, either by assuming a fast equilibration of the DNA component or by removing the RNA component. Here, stability of the fixed points of the three-component positive feedback loop is analyzed by obtaining eigenvalues of the full three-dimensional Hessian matrix. In addition to rigorously identifying the stable fixed points and saddle points, detailed information about the system can be obtained, such as the existence of complex eigenvalues near a fixed point.

  19. Analysis of proteins stained by Alexa dyes.

    PubMed

    Huang, Shijun; Wang, Houyi; Carroll, Christopher A; Hayes, Shirley J; Weintraub, Susan T; Serwer, Philip

    2004-03-01

    Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination. The Alexa-stained proteins are observed directly in gels by illumination with an ultraviolet transilluminator. The test multimolecular particle is bacteriophage T7. The protein capsid of T7 is a multimolecular complex that has both external and internal proteins. SDS-PAGE of Alexa-stained bacteriophage T7 produces fluorescent capsid proteins each of which usually comigrates with an unstained protein. However, one Alexa-induced modification of protein migration was observed by SDS-PAGE. Mass spectrometry shows that the protein with modified migration is the major protein of the outer shell of the T7 capsid. The procedures used are generally applicable. The distribution of Alexa staining among T7 capsid proteins depends on the size of the dye molecule used. The larger the dye molecule is, the greater the preference for external proteins.

  20. Handling of solid brain tumor tissue for protein analysis.

    PubMed

    Ericsson, Christer; Nistér, Monica

    2011-01-01

    Optimal protein analysis requires unfixed tissue samples. We suggest handling the brain tumor tissue sterilely and coldly (on ice) for as short time as possible prior to processing, but for no more than 8 h. This simple protocol results in apparently intact morphology, immunoreactivity, protein integrity, and protein phosphorylation with the criteria we apply. Sample handling for Pathological Anatomical Diagnosis (PAD) and for protein analysis can be one and the same.

  1. Sequence analysis of the AAA protein family.

    PubMed Central

    Beyer, A.

    1997-01-01

    The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases. PMID:9336829

  2. Protein detection by Simple Western™ analysis.

    PubMed

    Harris, Valerie M

    2015-01-01

    Protein Simple© has taken a well-known protein detection method, the western blot, and revolutionized it. The Simple Western™ system uses capillary electrophoresis to identify and quantitate a protein of interest. Protein Simple© provides multiple detection apparatuses (Wes, Sally Sue, or Peggy Sue) that are suggested to save scientists valuable time by allowing the researcher to prepare the protein sample, load it along with necessary antibodies and substrates, and walk away. Within 3-5 h the protein will be separated by size, or charge, immuno-detection of target protein will be accurately quantitated, and results will be immediately made available. Using the Peggy Sue instrument, one study recently examined changes in MAPK signaling proteins in the sex-determining stage of gonadal development. Here the methodology is described.

  3. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  4. ProteinHistorian: tools for the comparative analysis of eukaryote protein origin.

    PubMed

    Capra, John A; Williams, Alexander G; Pollard, Katherine S

    2012-01-01

    The evolutionary history of a protein reflects the functional history of its ancestors. Recent phylogenetic studies identified distinct evolutionary signatures that characterize proteins involved in cancer, Mendelian disease, and different ontogenic stages. Despite the potential to yield insight into the cellular functions and interactions of proteins, such comparative phylogenetic analyses are rarely performed, because they require custom algorithms. We developed ProteinHistorian to make tools for performing analyses of protein origins widely available. Given a list of proteins of interest, ProteinHistorian estimates the phylogenetic age of each protein, quantifies enrichment for proteins of specific ages, and compares variation in protein age with other protein attributes. ProteinHistorian allows flexibility in the definition of protein age by including several algorithms for estimating ages from different databases of evolutionary relationships. We illustrate the use of ProteinHistorian with three example analyses. First, we demonstrate that proteins with high expression in human, compared to chimpanzee and rhesus macaque, are significantly younger than those with human-specific low expression. Next, we show that human proteins with annotated regulatory functions are significantly younger than proteins with catalytic functions. Finally, we compare protein length and age in many eukaryotic species and, as expected from previous studies, find a positive, though often weak, correlation between protein age and length. ProteinHistorian is available through a web server with an intuitive interface and as a set of command line tools; this allows biologists and bioinformaticians alike to integrate these approaches into their analysis pipelines. ProteinHistorian's modular, extensible design facilitates the integration of new datasets and algorithms. The ProteinHistorian web server, source code, and pre-computed ages for 32 eukaryotic genomes are freely available under

  5. ProteinHistorian: Tools for the Comparative Analysis of Eukaryote Protein Origin

    PubMed Central

    Capra, John A.; Williams, Alexander G.; Pollard, Katherine S.

    2012-01-01

    The evolutionary history of a protein reflects the functional history of its ancestors. Recent phylogenetic studies identified distinct evolutionary signatures that characterize proteins involved in cancer, Mendelian disease, and different ontogenic stages. Despite the potential to yield insight into the cellular functions and interactions of proteins, such comparative phylogenetic analyses are rarely performed, because they require custom algorithms. We developed ProteinHistorian to make tools for performing analyses of protein origins widely available. Given a list of proteins of interest, ProteinHistorian estimates the phylogenetic age of each protein, quantifies enrichment for proteins of specific ages, and compares variation in protein age with other protein attributes. ProteinHistorian allows flexibility in the definition of protein age by including several algorithms for estimating ages from different databases of evolutionary relationships. We illustrate the use of ProteinHistorian with three example analyses. First, we demonstrate that proteins with high expression in human, compared to chimpanzee and rhesus macaque, are significantly younger than those with human-specific low expression. Next, we show that human proteins with annotated regulatory functions are significantly younger than proteins with catalytic functions. Finally, we compare protein length and age in many eukaryotic species and, as expected from previous studies, find a positive, though often weak, correlation between protein age and length. ProteinHistorian is available through a web server with an intuitive interface and as a set of command line tools; this allows biologists and bioinformaticians alike to integrate these approaches into their analysis pipelines. ProteinHistorian's modular, extensible design facilitates the integration of new datasets and algorithms. The ProteinHistorian web server, source code, and pre-computed ages for 32 eukaryotic genomes are freely available under

  6. Minimalist models for proteins: a comparative analysis.

    PubMed

    Tozzini, Valentina

    2010-08-01

    The last decade has witnessed a renewed interest in the coarse-grained (CG) models for biopolymers, also stimulated by the needs of modern molecular biology, dealing with nano- to micro-sized bio-molecular systems and larger than microsecond timescale. This combination of size and timescale is, in fact, hard to access by atomic-based simulations. Coarse graining the system is a route to be followed to overcome these limits, but the ways of practically implementing it are many and different, making the landscape of CG models very vast and complex. In this paper, the CG models are reviewed and their features, applications and performances compared. This analysis, restricted to proteins, focuses on the minimalist models, namely those reducing at minimum the number of degrees of freedom without losing the possibility of explicitly describing the secondary structures. This class includes models using a single or a few interacting centers (beads) for each amino acid. From this analysis several issues emerge. The difficulty in building these models resides in the need for combining transferability/predictive power with the capability of accurately reproducing the structures. It is shown that these aspects could be optimized by accurately choosing the force field (FF) terms and functional forms, and combining different parameterization procedures. In addition, in spite of the variety of the minimalist models, regularities can be found in the parameters values and in FF terms. These are outlined and schematically presented with the aid of a generic phase diagram of the polypeptide in the parameter space and, hopefully, could serve as guidelines for the development of minimalist models incorporating the maximum possible level of predictive power and structural accuracy.

  7. Structural Analysis of Proteins in Extreme Saline Environments

    DTIC Science & Technology

    1989-03-16

    for growth, obtain energy from amino acids, grow in media with high concentrations of protein hydrolysates , have very high intracellular salt...do )C/ ~(C N STRUCTURAL ANALYSIS OF PROTEINS IN EXTREME SALINE ENVIRONMENTS by 0 Dr. M. Shoham & Prof. 3. L. Sussman )IR5!Wcizmann Institute of...9 d) Charge Distribution on the Surface of the Protein ................................ 10 e) Adaptation to High Salt

  8. Screening serum hepatocellular carcinoma-associated proteins by SELDI-based protein spectrum analysis

    PubMed Central

    Cui, Jie-Feng; Liu, Yin-Kun; Zhou, Hai-Jun; Kang, Xiao-Nan; Huang, Cheng; He, Yi-Feng; Tang, Zhao-You; Uemura, Toshimasa

    2008-01-01

    AIM: To find out potential serum hepatocellular carcinoma (HCC)-associated proteins with low molecular weight and low abundance by SELDI-based serum protein spectra analysis, that will have much application in the diagnosis or differentiated diagnosis of HCC, as well as giving a better understanding of the mechanism of hepato-carcinogenesis. METHODS: Total serum samples were collected with informed consent from 81 HCC patients with HBV(+)/cirrhosis(+), 36 cirrhosis patients and 43 chronic hepatitis B patients. Serum protein fingerprint profiles were first generated by selected WCX2 protein chip capture integrating with SELDI-TOF-MS, then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard. Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra, some protein peaks with significant difference between HCC and cirrhosis or chronic hepatitis B were found. RESULTS: One hundred and twenty-eight serum protein peaks between 2000 and 30 000Da were identified under the condition of signal-to-noise > 5 and minimum threshold for cluster > 20%. Eighty-seven of these proteins were showed significant differences in intensity between HCC and cirrhosis (P < 0.05). Of the above differential proteins, 45 proteins had changes greater than two-fold, including 15 upregulated proteins and 30 downregulated proteins in HCC serum. Between HCC and chronic hepatitis B, 9 of 52 differential proteins (P < 0.05) had intensities of more than two-fold, including 2 upregulated proteins and 7 downregulated proteins in HCC serum. Between cirrhosis and chronic hepatitis B, 28 of 79 significant differential proteins (P < 0.05) changes greater than two-fold in intensity, including 17 upregulated proteins and 11 downregulated proteins in cirrhosis serum. For the analysis of these leading differential proteins in subtraction difference mode among three diseases, the five common downregulated proteins in HCC serum (M/Z 2870

  9. Screening serum hepatocellular carcinoma-associated proteins by SELDI-based protein spectrum analysis.

    PubMed

    Cui, Jie-Feng; Liu, Yin-Kun; Zhou, Hai-Jun; Kang, Xiao-Nan; Huang, Cheng; He, Yi-Feng; Tang, Zhao-You; Uemura, Toshimasa

    2008-02-28

    To find out potential serum hepatocellular carcinoma (HCC)-associated proteins with low molecular weight and low abundance by SELDI-based serum protein spectra analysis, that will have much application in the diagnosis or differentiated diagnosis of HCC, as well as giving a better understanding of the mechanism of hepato-carcinogenesis. Total serum samples were collected with informed consent from 81 HCC patients with HBV(+)/cirrhosis(+), 36 cirrhosis patients and 43 chronic hepatitis B patients. Serum protein fingerprint profiles were first generated by selected WCX2 protein chip capture integrating with SELDI-TOF-MS, then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard. Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra, some protein peaks with significant difference between HCC and cirrhosis or chronic hepatitis B were found. One hundred and twenty-eight serum protein peaks between 2000 and 30000 Da were identified under the condition of signal-to-noise > 5 and minimum threshold for cluster > 20%. Eighty-seven of these proteins were showed significant differences in intensity between HCC and cirrhosis (P < 0.05). Of the above differential proteins, 45 proteins had changes greater than two-fold, including 15 upregulated proteins and 30 downregulated proteins in HCC serum. Between HCC and chronic hepatitis B, 9 of 52 differential proteins (P < 0.05) had intensities of more than two-fold, including 2 upregulated proteins and 7 downregulated proteins in HCC serum. Between cirrhosis and chronic hepatitis B, 28 of 79 significant differential proteins (P < 0.05) changes greater than two-fold in intensity, including 17 upregulated proteins and 11 downregulated proteins in cirrhosis serum. For the analysis of these leading differential proteins in subtraction difference mode among three diseases, the five common downregulated proteins in HCC serum (M/Z 2870, 3941, 2688, 3165, 5483

  10. Large-scale proteomic analysis of membrane proteins

    SciTech Connect

    Ahram, Mamoun; Springer, David L.

    2004-10-01

    Proteomic analysis of membrane proteins is promising in identification of novel candidates as drug targets and/or disease biomarkers. Despite notable technological developments, obstacles related to extraction and solubilization of membrane proteins are frequently encountered. A critical discussion of the different preparative methods of membrane proteins is offered in relation to downstream proteomic applications, mainly gel-based analyses and mass spectrometry. Unknown proteins are often identified by high-throughput profiling of membrane proteins. In search for novel membrane proteins, analysis of protein sequences using computational tools is performed to predict for the presence of transmembrane domains. Here, we also present these bioinformatic tools with the human proteome as a case study. Along with technological innovations, advancements in the areas of sample preparation and computational prediction of membrane proteins will lead to exciting discoveries.

  11. APols-aided protein precipitation: a rapid method for concentrating proteins for proteomic analysis.

    PubMed

    Ning, Zhibin; Hawley, Brett; Seebun, Deeptee; Figeys, Daniel

    2014-10-01

    Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a "One-stop" single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols' protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.

  12. The thermodynamic analysis of weak protein interactions using sedimentation equilibrium

    PubMed Central

    Dolinska, Monika B.; Wingfield, Paul T.

    2014-01-01

    Proteins self-associate to form dimers and tetramers. Purified proteins are used to study the thermodynamics of protein interactions using the analytical ultracentrifuge. In this approach, monomer – dimer equilibrium constants are directly measured at various temperatures. Data analysis is used to derive thermodynamic parameters such as Gibbs free energy, enthalpy and entropy which can predict which major forces are involved in protein association. PMID:25081741

  13. Rapid visco analysis of food protein pastes

    USDA-ARS?s Scientific Manuscript database

    Whey protein isolate (WPI) powders are used in many formulations to boost nutrients. To predict the pasting behavior of proteins, WPI was tested under varying temperatures, using the Rapid-Visco-Analyzer (RVA), under pasting temperatures from 65 to 75 degrees'C, RVA speeds from 100 to 500 rpm, and ...

  14. Prioritizing disease candidate proteins in cardiomyopathy-specific protein-protein interaction networks based on "guilt by association" analysis.

    PubMed

    Li, Wan; Chen, Lina; He, Weiming; Li, Weiguo; Qu, Xiaoli; Liang, Binhua; Gao, Qianping; Feng, Chenchen; Jia, Xu; Lv, Yana; Zhang, Siya; Li, Xia

    2013-01-01

    The cardiomyopathies are a group of heart muscle diseases which can be inherited (familial). Identifying potential disease-related proteins is important to understand mechanisms of cardiomyopathies. Experimental identification of cardiomyophthies is costly and labour-intensive. In contrast, bioinformatics approach has a competitive advantage over experimental method. Based on "guilt by association" analysis, we prioritized candidate proteins involving in human cardiomyopathies. We first built weighted human cardiomyopathy-specific protein-protein interaction networks for three subtypes of cardiomyopathies using the known disease proteins from Online Mendelian Inheritance in Man as seeds. We then developed a method in prioritizing disease candidate proteins to rank candidate proteins in the network based on "guilt by association" analysis. It was found that most candidate proteins with high scores shared disease-related pathways with disease seed proteins. These top ranked candidate proteins were related with the corresponding disease subtypes, and were potential disease-related proteins. Cross-validation and comparison with other methods indicated that our approach could be used for the identification of potentially novel disease proteins, which may provide insights into cardiomyopathy-related mechanisms in a more comprehensive and integrated way.

  15. Emerging methods for structural analysis of protein aggregation.

    PubMed

    Khan, Eshan; Kumar, Amit

    2017-02-06

    Protein misfolding and aggregation is a key attribute of different neurodegenerative diseases. Misfolded and aggregated proteins are intrinsically disordered and rule out structure based drug design. The comprehensive characterization of misfolded proteins and associated aggregation pathway is prerequisite to develop therapeutics for neurodegenerative diseases caused due to the protein aggregation. Visible protein aggregates used to be the final stage during aggregation mechanism. The structural analysis of intermediate steps in such protein aggregates will help us to discern the conformational role and subsequently involved pathways. The structural analysis of protein aggregation using various biophysical methods may aid the improved therapeutics for protein misfolding and aggregation related neurodegenerative diseases. In this mini review, we have summarized different spectroscopic methods such as fluorescence spectroscopy, circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy for structural analysis of protein aggregation. We believe that the understanding of invisible intermediate of misfolded proteins and the key steps involved during protein aggregation mechanisms may advance the therapeutic approaches for targeting neurological diseases that are caused due to misfolded proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Computational analysis of maltose binding protein translocation

    NASA Astrophysics Data System (ADS)

    Chinappi, Mauro; Cecconi, Fabio; Massimo Casciola, Carlo

    2011-05-01

    We propose a computational model for the study of maltose binding protein translocation across α-hemolysin nanopores. The phenomenological approach simplifies both the pore and the polypeptide chain; however it retains the basic structural protein-like properties of the maltose binding protein by promoting the correct formation of its native key interactions. By considering different observables characterising the channel blockade and molecule transport, we verified that MD simulations reproduce qualitatively the behaviour observed in a recent experiment. Simulations reveal that blockade events consist of a capture stage, to some extent related to the unfolding kinetics, and a single file translocation process in the channel. A threshold mechanics underlies the process activation with a critical force depending on the protein denaturation state. Finally, our results support the simple interpretation of translocation via first-passage statistics of a driven diffusion process of a single reaction coordinate.

  17. Analysis of protein isoforms: can we do it better?

    PubMed

    Stastna, Miroslava; Van Eyk, Jennifer E

    2012-10-01

    Protein isoforms/splice variants can play important roles in various biological processes and can potentially be used as biomarkers or therapeutic targets/mediators. Thus, there is a need for efficient and, importantly, accurate methods to distinguish and quantify specific protein isoforms. Since protein isoforms can share a high percentage of amino acid sequence homology and dramatically differ in their cellular concentration, the task for accuracy and efficiency in methodology and instrumentation is challenging. The analysis of intact proteins has been perceived to provide a more accurate and complete result for isoform identification/quantification in comparison to analysis of the corresponding peptides that arise from protein enzymatic digestion. Recently, novel approaches have been explored and developed that can possess the accuracy and reliability important for protein isoform differentiation and isoform-specific peptide targeting. In this review, we discuss the recent development in methodology and instrumentation for enhanced detection of protein isoforms as well as the examples of their biological importance.

  18. Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins.

    PubMed

    Natarajan, Savithiry; Xu, Chenping; Caperna, Thomas J; Garrett, Wesley M

    2005-07-15

    Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods-urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone-to determine their efficacy in separating soybean seed proteins by 2D-PAGE. In all four methods, seed storage proteins were well separated by 2D-PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin, and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were beta-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.

  19. Protein synthesis in synaptosomes: a proteomics analysis.

    PubMed

    Jiménez, C R; Eyman, M; Lavina, Z Scotto; Gioio, A; Li, K W; van der Schors, R C; Geraerts, W P M; Giuditta, A; Kaplan, B B; van Minnen, J

    2002-05-01

    A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.

  20. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

    PubMed Central

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N. P.; Riedmayr, Lisa M.; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S.; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  1. Quantitative proteome analysis of colorectal cancer-related differential proteins.

    PubMed

    Zhang, Yanbin; Liu, Yue; Ye, Yingjiang; Shen, Danhua; Zhang, Hui; Huang, Hongyan; Li, Sha; Wang, Shan; Ren, Jun

    2017-02-01

    To evaluate a new strategy for profiling proteomic changes in colorectal cancer (CRC). We used laser capture microdissection (LCM) to obtain cells from 20 CRC and paired normal mucosal tissues. The differential proteins between the microdissected tumor cells and normal mucosa epithelia were analyzed by acetylation stable isotopic labeling coupled with L linear ion trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ-FT MS). Western blotting was used to assess the differential expression of proteins. We used bioinformatics tools for cluster and ingenuity pathway analysis of the differential proteins. In total, 798 confident proteins were quantified and 137 proteins were differentially expressed by at least twofold, including 67 that were upregulated and 70 that were downregulated in cancer. Two differential proteins, solute carrier family 12 member 2 (SLC12A2) and Ras-related protein Rab-10, were validated by Western blotting, and the results were consistent with acetylation stable isotopic labeling analysis. According to gene ontology analysis, CRC-related differential proteins covered a wide range of subcellular locations and were involved in many biological processes. According to ingenuity pathway analysis of the differential proteins, the most relevant canonical pathway associated with CRC was the 14-3-3-mediated signaling pathway, and seven reliable functional networks including cellular growth and proliferation, amino acid metabolism, inflammatory response, embryonic development, carbohydrate metabolism, cellular assembly and organization, and cell morphology were obtained. Combination of LCM, acetylation stable isotopic labeling analysis and LTQ-FT MS is effective for profiling proteomic changes in CRC cells.

  2. DNA mimic proteins: functions, structures, and bioinformatic analysis.

    PubMed

    Wang, Hao-Ching; Ho, Chun-Han; Hsu, Kai-Cheng; Yang, Jinn-Moon; Wang, Andrew H-J

    2014-05-13

    DNA mimic proteins have DNA-like negative surface charge distributions, and they function by occupying the DNA binding sites of DNA binding proteins to prevent these sites from being accessed by DNA. DNA mimic proteins control the activities of a variety of DNA binding proteins and are involved in a wide range of cellular mechanisms such as chromatin assembly, DNA repair, transcription regulation, and gene recombination. However, the sequences and structures of DNA mimic proteins are diverse, making them difficult to predict by bioinformatic search. To date, only a few DNA mimic proteins have been reported. These DNA mimics were not found by searching for functional motifs in their sequences but were revealed only by structural analysis of their charge distribution. This review highlights the biological roles and structures of 16 reported DNA mimic proteins. We also discuss approaches that might be used to discover new DNA mimic proteins.

  3. Serum proteins are extracted along with monolayer cells in plasticware and interfere with protein analysis

    PubMed Central

    Hong, Xin; Meng, Yuling; Kalkanis, Steven N.

    2016-01-01

    Washing and lysing monolayer cells directly from cell culture plasticware is a commonly used method for protein extraction. We found that multiple protein bands were enriched in samples with low cell numbers from the 6-well plate cultures. These proteins contributed to the overestimation of cell proteins and led to the uneven protein loading in Western blotting analysis. In Coomassie blue stained SDS-PAGE gels, the main enriched protein band is about 69 kDa and it makes up 13.6% of total protein from 104 U251n cells. Analyzed by mass spectrometry, we identified two of the enriched proteins: bovine serum albumin and bovine serum transferrin. We further observed that serum proteins could be extracted from other cell culture plates, dishes and flasks even after washing the cells 3 times with PBS. A total of 2.3 mg of protein was collected from a single well of the 6-well plate. A trace amount of the protein band was still visible after washing the cells 5 times with PBS. Thus, serum proteins should be considered if extracting proteins from plasticware, especially for samples with low cell numbers. PMID:27631018

  4. Topology analysis and visualization of Potyvirus protein-protein interaction network.

    PubMed

    Bosque, Gabriel; Folch-Fortuny, Abel; Picó, Jesús; Ferrer, Alberto; Elena, Santiago F

    2014-11-20

    One of the central interests of Virology is the identification of host factors that contribute to virus infection. Despite tremendous efforts, the list of factors identified remains limited. With omics techniques, the focus has changed from identifying and thoroughly characterizing individual host factors to the simultaneous analysis of thousands of interactions, framing them on the context of protein-protein interaction networks and of transcriptional regulatory networks. This new perspective is allowing the identification of direct and indirect viral targets. Such information is available for several members of the Potyviridae family, one of the largest and more important families of plant viruses. After collecting information on virus protein-protein interactions from different potyviruses, we have processed it and used it for inferring a protein-protein interaction network. All proteins are connected into a single network component. Some proteins show a high degree and are highly connected while others are much less connected, with the network showing a significant degree of dissortativeness. We have attempted to integrate this virus protein-protein interaction network into the largest protein-protein interaction network of Arabidopsis thaliana, a susceptible laboratory host. To make the interpretation of data and results easier, we have developed a new approach for visualizing and analyzing the dynamic spread on the host network of the local perturbations induced by viral proteins. We found that local perturbations can reach the entire host protein-protein interaction network, although the efficiency of this spread depends on the particular viral proteins. By comparing the spread dynamics among viral proteins, we found that some proteins spread their effects fast and efficiently by attacking hubs in the host network while other proteins exert more local effects. Our findings confirm that potyvirus protein-protein interaction networks are highly connected, with

  5. Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.

    PubMed

    Pawlak, Michael; Schick, Eginhard; Bopp, Martin A; Schneider, Michael J; Oroszlan, Peter; Ehrat, Markus

    2002-04-01

    Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will

  6. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  7. Dynamic network analysis of protein interactions

    NASA Astrophysics Data System (ADS)

    Almaas, Eivind; Deri, Joya

    2007-03-01

    Network approaches have recently become a popular tool to study complex systems such as cellular metabolism and protein interactions. A substantial number of analyses of the protein interaction network (PIN) of the yeast Saccharomyces cerevisiae have considered this network as a static entity, not taking the network's dynamic nature into account. Here, we examine the time-variation of gene regulation superimposed on the PIN by defining mRNA expression profiles throughout the cell cycle as node weights. To characterize these network dynamics, we have both developed a set of novel network measures as well as studied previously published measures for weighted networks. We expect that our approach will provide a deeper understanding of protein regulation during the cell cycle.

  8. Analysis of HIV Proteins Using DSP Techniques

    DTIC Science & Technology

    2007-11-02

    envelope protein, gp160 is initially divided into two fragments proteins gp120 and gp41 , which then bind to each other to interact with the CD4 cell...envelope revealed three peptides: DP-107, peptide-637- 666 and the most potent, T20, all from gp41 have antiviral activity [5-7]. The mechanism by which...19 HIV-1 isolates indeed have one common frequency f=0.185+0.001 (Fig.1). The same frequency was identified as common for gp41 and gp120

  9. Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

    PubMed

    Bengali, Aditya N; Tessier, Peter M

    2009-10-01

    "Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these

  10. Introduction of inflammatory bowel disease biomarkers panel using protein-protein interaction (PPI) network analysis.

    PubMed

    Asadzadeh-Aghdaee, Hamid; Shahrokh, Shabnam; Norouzinia, Mohsen; Hosseini, Mostafa; Keramatinia, Aliasghar; Jamalan, Mostafa; Naghibzadeh, Bijan; Sadeghi, Ali; Jahani Sherafat, Somayeh; Zali, Mohammad Reza

    2016-12-01

    In the present study, a protein-protein interaction network construction is conducted for IBD. Inflammatory bowel diseases as serious chronic gastrointestinal disorders attracted many molecular investigations. Diverse molecular information is present for IBD. However, these molecular findings are not highlighted based on interactome analysis. On the other hand, PPI network analysis is a powerful method for study of molecular interactions in the protein level that provide useful information for highlighting the desired key proteins. Cytoscape is the used software with its plug-ins for detailed analysis. Two centrality parameters including degree and betweenness are determined and the crucial proteins based on these parameters are introduced. The 75 proteins among 100 initial proteins are included in the network of IBD. Seventy-five nodes and 260 edges constructed the network as a scale free network. The findings indicate that there are seven hub-bottleneck proteins in the IBD network. More examination revealed the essential roles of these key proteins in the integrity of the network. Finally, the indicator panel including NFKB1, CD40, TNFA, TYK2, NOD2, IL23R, and STAT3 is presented as a possible molecular index for IBD.

  11. Introduction of inflammatory bowel disease biomarkers panel using protein-protein interaction (PPI) network analysis

    PubMed Central

    Asadzadeh-Aghdaee, Hamid; Shahrokh, Shabnam; Norouzinia, Mohsen; Hosseini, Mostafa; Keramatinia, Aliasghar; Jamalan, Mostafa; Naghibzadeh, Bijan; Sadeghi, Ali; Jahani Sherafat, Somayeh; Zali, Mohammad Reza

    2016-01-01

    Aim: In the present study, a protein-protein interaction network construction is conducted for IBD. Background: Inflammatory bowel diseases as serious chronic gastrointestinal disorders attracted many molecular investigations. Diverse molecular information is present for IBD. However, these molecular findings are not highlighted based on interactome analysis. On the other hand, PPI network analysis is a powerful method for study of molecular interactions in the protein level that provide useful information for highlighting the desired key proteins. Methods: Cytoscape is the used software with its plug-ins for detailed analysis. Two centrality parameters including degree and betweenness are determined and the crucial proteins based on these parameters are introduced. Results: The 75 proteins among 100 initial proteins are included in the network of IBD. Seventy-five nodes and 260 edges constructed the network as a scale free network. The findings indicate that there are seven hub-bottleneck proteins in the IBD network. Conclusion: More examination revealed the essential roles of these key proteins in the integrity of the network. Finally, the indicator panel including NFKB1, CD40, TNFA, TYK2, NOD2, IL23R, and STAT3 is presented as a possible molecular index for IBD. PMID:28224022

  12. SCOWLP classification: Structural comparison and analysis of protein binding regions

    PubMed Central

    Teyra, Joan; Paszkowski-Rogacz, Maciej; Anders, Gerd; Pisabarro, M Teresa

    2008-01-01

    Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs) might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions. The hierarchical

  13. Protein Analysis-on-Chip Systems in Foodomics

    PubMed Central

    Nazzaro, Filomena; Orlando, Pierangelo; Fratianni, Florinda; Di Luccia, Aldo; Coppola, Raffaele

    2012-01-01

    Protein compositional data can address nutritional, packaging, origin/authenticity, processing history, safety and other quality questions. Such data has been time-consuming and expensive to generate until recently but “protein analysis on a chip” systems are now available that can analyze a complex food sample in a few minutes and do not require great protein analytical expertise. We review some of the main new approaches with examples of their application and discuss their advantages and disadvantages. PMID:23201766

  14. Protein analysis-on-chip systems in foodomics.

    PubMed

    Nazzaro, Filomena; Orlando, Pierangelo; Fratianni, Florinda; Di Luccia, Aldo; Coppola, Raffaele

    2012-10-16

    Protein compositional data can address nutritional, packaging, origin/authenticity, processing history, safety and other quality questions. Such data has been time-consuming and expensive to generate until recently but "protein analysis on a chip" systems are now available that can analyze a complex food sample in a few minutes and do not require great protein analytical expertise. We review some of the main new approaches with examples of their application and discuss their advantages and disadvantages.

  15. Proteomics analysis of egg white proteins from different egg varieties.

    PubMed

    Wang, Jiapei; Liang, Yue; Omana, Dileep A; Kav, Nat N V; Wu, Jianping

    2012-01-11

    The market of specialty eggs, such as omega-3-enriched eggs, organic eggs, and free-range eggs, is continuously growing. The nutritional composition of egg yolk can be manipulated by feed diet; however, it is not known if there is any difference in the composition of egg white proteins among different egg varieties. The purpose of the study was to compare the egg white proteins among six different egg varieties using proteomics analysis. Egg white proteins were analyzed using two-dimensional gel electrophoresis (2-DE), and 89 protein spots were subjected to LC-MS/MS. A total of 23 proteins, belonging to Gallus gallus , were identified from 72 detected protein spots. A quiescence-specific protein precursor in egg white was identified for the first time in this study. Significant differences in the abundant levels of 19 proteins (from 65 protein spots) were observed among six egg varieties. Four proteins, ovalbumin-related protein Y, cystatin, avidin, and albumin precursor, were not different among these six egg varieties. These findings suggest that the abundance, but not the composition, of egg white proteins varied among the egg varieties.

  16. Sodium loading changes urinary protein excretion: a proteomic analysis.

    PubMed

    Thongboonkerd, Visith; Klein, Jon B; Pierce, William M; Jevans, Anthony W; Arthur, John M

    2003-06-01

    Plasma sodium concentration is maintained even when sodium intake is altered. Sodium homeostasis may involve changes in renal tubular protein expression that are reflected in the urine. We used proteomic analysis to investigate changes in urinary protein excretion in response to acute sodium loading. Rats were given deionized water followed by hypertonic (2.7%) saline for 28 h each. Urinary protein expression was determined during the final 4 h of each treatment. Acute sodium loading increased urinary sodium excretion (4.53 +/- 1.74 vs. 1.70 +/- 0.27 mmol/day, P = 0.029). Urinary proteins were separated by two-dimensional PAGE and visualized by Sypro ruby staining. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry followed by peptide mass fingerprinting. The abundance of a total of 45 protein components was changed after acute sodium loading. Neutral endopeptidase, solute carrier family 3, meprin 1alpha, diphor-1, chaperone heat shock protein 72, vacuolar H(+)-ATPase, ezrin, ezrin/radixin/moesin-binding protein, glutamine synthetase, guanine nucleotide-binding protein, Rho GDI-1, and chloride intracellular channel protein 1 were decreased, whereas albumin and alpha-2u globulin were increased. Some of these proteins have previously been shown to be associated with tubular transport. These data indicate that alterations in the excretion of several urinary proteins occur during acute sodium loading.

  17. Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins

    PubMed Central

    Zhao, Liming; Huang, Yong; Hu, Yan; He, Xiaoli; Shen, Wenhui; Liu, Chunlin; Ruan, Ying

    2013-01-01

    The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel β-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa PMID:24179444

  18. An analysis pipeline for the inference of protein-protein interaction networks

    SciTech Connect

    Taylor, Ronald C.; Singhal, Mudita; Daly, Don S.; Gilmore, Jason M.; Cannon, William R.; Domico, Kelly O.; White, Amanda M.; Auberry, Deanna L.; Auberry, Kenneth J.; Hooker, Brian S.; Hurst, G. B.; McDermott, Jason E.; McDonald, W. H.; Pelletier, Dale A.; Schmoyer, Denise A.; Wiley, H. S.

    2009-12-01

    An analysis pipeline has been created for deployment of a novel algorithm, the Bayesian Estimator of Protein-Protein Association Probabilities (BEPro), for use in the reconstruction of protein-protein interaction networks. We have combined the Software Environment for BIological Network Inference (SEBINI), an interactive environment for the deployment and testing of network inference algorithms that use high-throughput data, and the Collective Analysis of Biological Interaction Networks (CABIN), software that allows integration and analysis of protein-protein interaction and gene-to-gene regulatory evidence obtained from multiple sources, to allow interactions computed by BEPro to be stored, visualized, and further analyzed. Incorporating BEPro into SEBINI and automatically feeding the resulting inferred network into CABIN, we have created a structured workflow for protein-protein network inference and supplemental analysis from sets of mass spectrometry bait-prey experiment data. SEBINI demo site: https://www.emsl.pnl.gov /SEBINI/ Contact: ronald.taylor@pnl.gov. BEPro is available at http://www.pnl.gov/statistics/BEPro3/index.htm. Contact: ds.daly@pnl.gov. CABIN is available at http://www.sysbio.org/dataresources/cabin.stm. Contact: mudita.singhal@pnl.gov.

  19. An analysis pipeline for the inferenceof protein-protein interaction networks

    SciTech Connect

    Taylor, Ronald C.; Singhal, Mudita; Daly, Don S.; Gilmore, Jason; Cannon, Bill; Domico, Kelly; White, Amanda M.; Auberry, Deanna L; Auberry, Kenneth J; Hooker, Brian; Hurst, Gregory {Greg} B; McDermott, Jason; McDonald, W Hayes; Pelletier, Dale A; Schmoyer, Denise D; Wiley, Steven

    2009-09-01

    We present an integrated platform that is used for the reconstruction and analysis of protein-protein interaction networks inferred from Mass Spectrometry (MS) bait-prey experiment data. At the heart of this pipeline is the Software Environment for Biological Network Inference (SEBINI), an interactive environment for the deployment and testing of network inference algorithms that use high-throughput data. Among the many algorithms available in SEBINI is the Bayesian Estimator of Probabilities of Protein-Protein Associations (BEPro3) algorithm, which is used to infer interaction networks from such MS affinity isolation data. For integration, comparison and analysis of the inferred protein-protein interactions with interaction evidence obtained from multiple public sources, the pipeline connects to the Collective Analysis of Biological Interaction Networks (CABIN) software. Incorporating BEPro3 into SEBINI and automatically feeding the resulting inferred network into CABIN, we have created a structured workflow for protein-protein network inference and supplemental analysis from sets of MS bait-prey experiments.

  20. Transgenic soybeans and soybean protein analysis: an overview.

    PubMed

    Natarajan, Savithiry; Luthria, Devanand; Bae, Hanhong; Lakshman, Dilip; Mitra, Amitava

    2013-12-04

    To meet the increasing global demand for soybeans for food and feed consumption, new high-yield varieties with improved quality traits are needed. To ensure the safety of the crop, it is important to determine the variation in seed proteins along with unintended changes that may occur in the crop as a result various stress stimuli, breeding, and genetic modification. Understanding the variation of seed proteins in the wild and cultivated soybean cultivars is useful for determining unintended protein expression in new varieties of soybeans. Proteomic technology is useful to analyze protein variation due to various stimuli. This short review discusses transgenic soybeans, different soybean proteins, and the approaches used for protein analysis. The characterization of soybean protein will be useful for researchers, nutrition professionals, and regulatory agencies dealing with soy-derived food products.

  1. Protein conjugation with PAMAM nanoparticles: Microscopic and thermodynamic analysis.

    PubMed

    Chanphai, P; Froehlich, E; Mandeville, J S; Tajmir-Riahi, H A

    2017-02-01

    PAMAM dendrimers form strong protein conjugates that are used in drug delivery systems. We report the thermodynamic and binding analysis of polyamidoamine (PAMAM-G4) conjugation with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. Hydrophobicity played a major role in PAMAM-protein interactions with more hydrophobic b-LG forming stronger polymer-protein conjugates. Thermodynamic parameters showed PAMAM-protein bindings occur via hydrophobic and H-bonding contacts for b-LG, while van der waals and H-bonding interactions prevail in HSA and BSA-polymer conjugates. The protein loading efficacy was 45-55%. PAMAM complexation induced major alterations of protein conformation. TEM images show major polymer morphological changes upon protein conjugation.

  2. ComPPI: a cellular compartment-specific database for protein-protein interaction network analysis.

    PubMed

    Veres, Daniel V; Gyurkó, Dávid M; Thaler, Benedek; Szalay, Kristóf Z; Fazekas, Dávid; Korcsmáros, Tamás; Csermely, Peter

    2015-01-01

    Here we present ComPPI, a cellular compartment-specific database of proteins and their interactions enabling an extensive, compartmentalized protein-protein interaction network analysis (URL: http://ComPPI.LinkGroup.hu). ComPPI enables the user to filter biologically unlikely interactions, where the two interacting proteins have no common subcellular localizations and to predict novel properties, such as compartment-specific biological functions. ComPPI is an integrated database covering four species (S. cerevisiae, C. elegans, D. melanogaster and H. sapiens). The compilation of nine protein-protein interaction and eight subcellular localization data sets had four curation steps including a manually built, comprehensive hierarchical structure of >1600 subcellular localizations. ComPPI provides confidence scores for protein subcellular localizations and protein-protein interactions. ComPPI has user-friendly search options for individual proteins giving their subcellular localization, their interactions and the likelihood of their interactions considering the subcellular localization of their interacting partners. Download options of search results, whole-proteomes, organelle-specific interactomes and subcellular localization data are available on its website. Due to its novel features, ComPPI is useful for the analysis of experimental results in biochemistry and molecular biology, as well as for proteome-wide studies in bioinformatics and network science helping cellular biology, medicine and drug design.

  3. A proteomic strategy for global analysis of plant protein complexes.

    PubMed

    Aryal, Uma K; Xiong, Yi; McBride, Zachary; Kihara, Daisuke; Xie, Jun; Hall, Mark C; Szymanski, Daniel B

    2014-10-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

  4. Comprehensive Proteomic Analysis of Membrane Proteins in Toxoplasma gondii*

    PubMed Central

    Che, Fa-Yun; Madrid-Aliste, Carlos; Burd, Berta; Zhang, Hongshan; Nieves, Edward; Kim, Kami; Fiser, Andras; Angeletti, Ruth Hogue; Weiss, Louis M.

    2011-01-01

    Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer “sandwich” gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies. PMID:20935347

  5. Large scale analysis of protein stability in OMIM disease related human protein variants.

    PubMed

    Martelli, Pier Luigi; Fariselli, Piero; Savojardo, Castrense; Babbi, Giulia; Aggazio, Francesco; Casadio, Rita

    2016-06-23

    Modern genomic techniques allow to associate several Mendelian human diseases to single residue variations in different proteins. Molecular mechanisms explaining the relationship among genotype and phenotype are still under debate. Change of protein stability upon variation appears to assume a particular relevance in annotating whether a single residue substitution can or cannot be associated to a given disease. Thermodynamic properties of human proteins and of their disease related variants are lacking. In the present work, we take advantage of the available three dimensional structure of human proteins for predicting the role of disease related variations on the perturbation of protein stability. We develop INPS3D, a new predictor based on protein structure for computing the effect of single residue variations on protein stability (ΔΔG), scoring at the state-of-the-art (Pearson's correlation value of the regression is equal to 0.72 with mean standard error of 1.15 kcal/mol on a blind test set comprising 351 variations in 60 proteins). We then filter 368 OMIM disease related proteins known with atomic resolution (where the three dimensional structure covers at least 70 % of the sequence) with 4717 disease related single residue variations and 685 polymorphisms without clinical consequence. We find that the effect on protein stability of disease related variations is larger than the effect of polymorphisms: in particular, by setting to |1 kcal/mol| the threshold between perturbing and not perturbing variations of the protein stability, about 44 % of disease related variations and 20 % of polymorphisms are predicted with |ΔΔG| > 1 kcal/mol, respectively. A consistent fraction of OMIM disease related variations is however predicted to promote |ΔΔG| ≤ 1 kcal/mol and we focus here on detecting features that can be associated to the thermodynamic property of the protein variant. Our analysis reveals that some 47 % of disease related variations

  6. Protein interface classification by evolutionary analysis

    PubMed Central

    2012-01-01

    Background Distinguishing biologically relevant interfaces from lattice contacts in protein crystals is a fundamental problem in structural biology. Despite efforts towards the computational prediction of interface character, many issues are still unresolved. Results We present here a protein-protein interface classifier that relies on evolutionary data to detect the biological character of interfaces. The classifier uses a simple geometric measure, number of core residues, and two evolutionary indicators based on the sequence entropy of homolog sequences. Both aim at detecting differential selection pressure between interface core and rim or rest of surface. The core residues, defined as fully buried residues (>95% burial), appear to be fundamental determinants of biological interfaces: their number is in itself a powerful discriminator of interface character and together with the evolutionary measures it is able to clearly distinguish evolved biological contacts from crystal ones. We demonstrate that this definition of core residues leads to distinctively better results than earlier definitions from the literature. The stringent selection and quality filtering of structural and sequence data was key to the success of the method. Most importantly we demonstrate that a more conservative selection of homolog sequences - with relatively high sequence identities to the query - is able to produce a clearer signal than previous attempts. Conclusions An evolutionary approach like the one presented here is key to the advancement of the field, which so far was missing an effective method exploiting the evolutionary character of protein interfaces. Its coverage and performance will only improve over time thanks to the incessant growth of sequence databases. Currently our method reaches an accuracy of 89% in classifying interfaces of the Ponstingl 2003 datasets and it lends itself to a variety of useful applications in structural biology and bioinformatics. We made the

  7. Molecular Analysis of Protein Assembly in Muscle Development.

    ERIC Educational Resources Information Center

    Epstein, Henry F.; Fischman, Donald A.

    1991-01-01

    Advances in the genetics and cell biology of muscle development are discussed. In-vitro analysis of the renaturation, polymerization, and three-dimensional structure of the purified proteins involved is described. (CW)

  8. Molecular Analysis of Protein Assembly in Muscle Development.

    ERIC Educational Resources Information Center

    Epstein, Henry F.; Fischman, Donald A.

    1991-01-01

    Advances in the genetics and cell biology of muscle development are discussed. In-vitro analysis of the renaturation, polymerization, and three-dimensional structure of the purified proteins involved is described. (CW)

  9. Surface energetics and protein-protein interactions: analysis and mechanistic implications

    NASA Astrophysics Data System (ADS)

    Peri, Claudio; Morra, Giulia; Colombo, Giorgio

    2016-04-01

    Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions.

  10. MESSA: MEta-Server for protein Sequence Analysis

    PubMed Central

    2012-01-01

    Background Computational sequence analysis, that is, prediction of local sequence properties, homologs, spatial structure and function from the sequence of a protein, offers an efficient way to obtain needed information about proteins under study. Since reliable prediction is usually based on the consensus of many computer programs, meta-severs have been developed to fit such needs. Most meta-servers focus on one aspect of sequence analysis, while others incorporate more information, such as PredictProtein for local sequence feature predictions, SMART for domain architecture and sequence motif annotation, and GeneSilico for secondary and spatial structure prediction. However, as predictions of local sequence properties, three-dimensional structure and function are usually intertwined, it is beneficial to address them together. Results We developed a MEta-Server for protein Sequence Analysis (MESSA) to facilitate comprehensive protein sequence analysis and gather structural and functional predictions for a protein of interest. For an input sequence, the server exploits a number of select tools to predict local sequence properties, such as secondary structure, structurally disordered regions, coiled coils, signal peptides and transmembrane helices; detect homologous proteins and assign the query to a protein family; identify three-dimensional structure templates and generate structure models; and provide predictive statements about the protein's function, including functional annotations, Gene Ontology terms, enzyme classification and possible functionally associated proteins. We tested MESSA on the proteome of Candidatus Liberibacter asiaticus. Manual curation shows that three-dimensional structure models generated by MESSA covered around 75% of all the residues in this proteome and the function of 80% of all proteins could be predicted. Availability MESSA is free for non-commercial use at http://prodata.swmed.edu/MESSA/ PMID:23031578

  11. Protein interaction discovery using parallel analysis of translated ORFs (PLATO).

    PubMed

    Zhu, Jian; Larman, H Benjamin; Gao, Geng; Somwar, Romel; Zhang, Zijuan; Laserson, Uri; Ciccia, Alberto; Pavlova, Natalya; Church, George; Zhang, Wei; Kesari, Santosh; Elledge, Stephen J

    2013-04-01

    Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method for human proteins by identifying known and previously unidentified interacting partners of LYN kinase, patient autoantibodies, and the small-molecules gefitinib and dasatinib.

  12. From quantitative protein complex analysis to disease mechanism.

    PubMed

    Texier, Y; Kinkl, N; Boldt, K; Ueffing, M

    2012-12-15

    Interest in the field of cilia biology and cilia-associated diseases - ciliopathies - has strongly increased over the last few years. Proteomic technologies, especially protein complex analysis by affinity purification-based methods, have been used to decipher various basic but also disease-associated mechanisms. This review focusses on some selected recent studies using affinity purification-based protein complex analysis, thereby exemplifying the great possibilities this technology offers.

  13. Advances in protein complex analysis using mass spectrometry

    PubMed Central

    Gingras, Anne-Claude; Aebersold, Ruedi; Raught, Brian

    2005-01-01

    Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein–protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)-based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal-to-noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented. PMID:15611014

  14. Persistent homology analysis of protein structure, flexibility and folding

    PubMed Central

    Xia, Kelin; Wei, Guo-Wei

    2014-01-01

    Proteins are the most important biomolecules for living organisms. The understanding of protein structure, function, dynamics and transport is one of most challenging tasks in biological science. In the present work, persistent homology is, for the first time, introduced for extracting molecular topological fingerprints (MTFs) based on the persistence of molecular topological invariants. MTFs are utilized for protein characterization, identification and classification. The method of slicing is proposed to track the geometric origin of protein topological invariants. Both all-atom and coarse-grained representations of MTFs are constructed. A new cutoff-like filtration is proposed to shed light on the optimal cutoff distance in elastic network models. Based on the correlation between protein compactness, rigidity and connectivity, we propose an accumulated bar length generated from persistent topological invariants for the quantitative modeling of protein flexibility. To this end, a correlation matrix based filtration is developed. This approach gives rise to an accurate prediction of the optimal characteristic distance used in protein B-factor analysis. Finally, MTFs are employed to characterize protein topological evolution during protein folding and quantitatively predict the protein folding stability. An excellent consistence between our persistent homology prediction and molecular dynamics simulation is found. This work reveals the topology-function relationship of proteins. PMID:24902720

  15. BIMOLECULAR FLUORESCENCE COMPLEMENTATION ANALYSIS OF INDUCIBLE PROTEIN INTERACTIONS: EFFECTS OF FACTORS AFFECTING PROTEIN FOLDING ON FLUORESCENT PROTEIN FRAGMENT ASSOCIATION

    PubMed Central

    Robida, Aaron M; Kerppola, Tom K

    2009-01-01

    Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. We investigated the temporal resolution and the quantitative accuracy of BiFC analysis using fragments of different fluorescent proteins. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of YFP fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 minutes after rapamycin addition and a ten-fold increase in the mean fluorescence intensity in 8 hours. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and YFP produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment of the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized prior to rapamycin addition formed BiFC complexes with the same efficiency as newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggest that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular

  16. Genome wide analysis of protein production load in Trichoderma reesei.

    PubMed

    Pakula, Tiina M; Nygren, Heli; Barth, Dorothee; Heinonen, Markus; Castillo, Sandra; Penttilä, Merja; Arvas, Mikko

    2016-01-01

    The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is a widely used industrial host organism for protein production. In industrial cultivations, it can produce over 100 g/l of extracellular protein, mostly constituting of cellulases and hemicellulases. In order to improve protein production of T. reesei the transcriptional regulation of cellulases and secretory pathway factors have been extensively studied. However, the metabolism of T. reesei under protein production conditions has not received much attention. To understand the physiology and metabolism of T. reesei under protein production conditions we carried out a well-controlled bioreactor experiment with extensive analysis. We used minimal media to make the data amenable for modelling and three strain pairs to cover different protein production levels. With RNA-sequencing transcriptomics we detected the concentration of the carbon source as the most important determinant of the transcriptome. As the major transcriptional response concomitant to protein production we detected the induction of selected genes that were putatively regulated by xyr1 and were related to protein transport, amino acid metabolism and transcriptional regulation. We found novel metabolic responses such as production of glycerol and a cellotriose-like compound. We then used this cultivation data for flux balance analysis of T. reesei metabolism and demonstrate for the first time the use of genome wide stoichiometric metabolic modelling for T. reesei. We show that our model can predict protein production rate and provides novel insight into the metabolism of protein production. We also provide this unprecedented cultivation and transcriptomics data set for future modelling efforts. The use of stoichiometric modelling can open a novel path for the improvement of protein production in T. reesei. Based on this we propose sulphur assimilation as a major limiting factor of protein production. As an organism with

  17. Analysis of protein dynamics using local-DME calculations.

    PubMed

    Wu, Di; Smith, Stephen; Mahan, Hannah; Jernigan, Robert L

    2011-01-01

    Flexibility and dynamics of protein structures are reflected in the B-factors and order parameters obtained experimentally with X-ray crystallography and Nuclear Magnetic Resonance (NMR). Methods such as Normal Mode Analysis (NMA) and Elastic Network Models (ENM) can be used to predict the fluctuations of protein structures for either atomic level or coarse-grained structures. Here, we introduce the Local-Distance Matrix Error (DME), an efficient and simple analytic method to study the fluctuations of protein structures, especially for the ensembles of NMR-determined protein structures. Comparisons with the fluctuations obtained by experiments and other by computations show strong correlations.

  18. Skeleton-based shape analysis of protein models.

    PubMed

    Li, Zhong; Qin, Shengwei; Yu, Zeyun; Jin, Yao

    2014-09-01

    In order to compare the similarity between two protein models, a shape analysis algorithm based on skeleton extraction is presented in this paper. It firstly extracts the skeleton of a given protein surface by an improved Multi-resolution Reeb Graph (MRG) method. A number of points on the model surface are then collected to compute the local diameter (LD) according to the skeleton. Finally the LD frequency is calculated to build up the line chart, which is employed to analyze the shape similarity between protein models. Experimental results show that the similarity comparison using the proposed shape descriptor is more accurate especially for protein models with large deformations.

  19. Purification of IFT particle proteins and preparation of recombinant proteins for structural and functional analysis.

    PubMed

    Behal, Robert H; Betleja, Ewelina; Cole, Douglas G

    2009-01-01

    Intraflagellar transport (IFT) is characterized by a robust bidirectional movement of large proteinaceous particles along the length of eukaryotic cilia and flagella. Essential for the assembly and function of the organelle, IFT is believed to transport a large array of ciliary components in and out of the organelle. Biochemical analysis of the proteins involved with this transport has been largely dependent on the ability to isolate suitable quantities of intact cilia or flagella. One model organism, Chlamydomonas reinhardtii, has proven to be especially well-suited for such endeavors. Indeed, many of the IFT particle proteins were initially identified through biochemical analysis of green algae. This chapter describes some of the most effective methods for the purification of IFT particle proteins from Chlamydomonas flagella. This chapter also describes complementary approaches where recombinant IFT proteins are generated with affinity tags that allow rapid and specific purification. The recombinant proteins can be used to analyze protein-protein interactions and can be directly delivered to mutant cells to analyze functional domains. Although the techniques described here are focused entirely on Chlamydomonas IFT proteins, the approaches, especially regarding recombinant proteins, should be applicable to the study of IFT machinery in other model organisms.

  20. Phthalic Acid Chemical Probes Synthesized for Protein-Protein Interaction Analysis

    PubMed Central

    Liang, Shih-Shin; Liao, Wei-Ting; Kuo, Chao-Jen; Chou, Chi-Hsien; Wu, Chin-Jen; Wang, Hui-Min

    2013-01-01

    Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES–SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis. PMID:23797655

  1. Analysis of Unfolded Protein Response in Arabidopsis

    PubMed Central

    Chen, Yani; Brandizzi, Federica

    2014-01-01

    The unfolded protein response (UPR) is fundamental for development and adaption in eukaryotic cells. Arabidopsis has become one of the best model systems to uncover conserved mechanisms of the UPR in multicellular eukaryotes as well as organism-specific regulation of the UPR in plants. Monitoring the UPR in planta is an elemental approach to identifying regulatory components and to revealing molecular mechanisms of the plant UPR. In this chapter, we provide protocols for the induction and analyses of plant UPR at a molecular level in Arabidopsis. Three kinds of ER stress treatment methods and quantitation of the plant UPR activation are described here. PMID:23913037

  2. 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.

  3. Analysis of proteins and peptides by electromigration methods in microchips.

    PubMed

    Štěpánová, Sille; Kašička, Václav

    2017-01-01

    This review presents the developments and applications of microchip electromigration methods in the separation and analysis of peptides and proteins in the period 2011-mid-2016. The developments in sample preparation and preconcentration, microchannel material, and surface treatment are described. Separations by various microchip electromigration methods (zone electrophoresis in free and sieving media, affinity electrophoresis, isotachophoresis, isoelectric focusing, electrokinetic chromatography, and electrochromatography) are demonstrated. Advances in detection methods are reported and novel applications in the areas of proteomics and peptidomics, quality control of peptide and protein pharmaceuticals, analysis of proteins and peptides in biomatrices, and determination of physicochemical parameters are shown.

  4. Analysis of protein composition using multidimensional chromatography and mass spectrometry.

    PubMed

    Link, Andrew J; Washburn, Michael P

    2014-11-03

    Multidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in-gel digestions by directly identifying protein mixtures in solution. One method used extensively is named Multidimensional Protein Identification Technology (MudPIT), where reversed-phase chromatography and strong cation-exchange chromatography are coupled directly in a microcapillary column. This column is then placed in line between an HPLC and a mass spectrometer for complex mixture analysis. MudPIT remains a powerful approach for analyzing complex mixtures like whole proteomes and protein complexes. MudPIT is used for quantitative proteomic analysis of complex mixtures to generate novel biological insights.

  5. Isolation and mass spectrometry analysis of urinary extraexosomal proteins

    PubMed Central

    Hildonen, Siri; Skarpen, Ellen; Halvorsen, Trine Grønhaug; Reubsaet, Léon

    2016-01-01

    The aim of the present study was to develop a LC-MS/MS-based proteomic analysis method of urinary exosomal proteins that has the potential to discover disease biomarkers. In short, urinary exosomes from healthy subjects were isolated by immunocapture on magnetic beads, detected by immunofluorescence and TEM, trypsin digested directly on the beads for an accelerated time with no addition of detergents before performing an LC-MS analysis of the trypsinate. To our knowledge, this is the first proteomic analysis of proteins displayed on the outer surface of exosomes. The outer exosome proteome may contain proteins that are of higher biomarker value compared to soluble cargo protein as the proteins projecting into the extracellular milieu might be more directly involved in physiological functions of exosomes. The proteomic analysis identified 49 proteins that were considered significant; the majority is involved in carbohydrate and lipid metabolism or in immune responses. Thirty of the proteins are linked to diseases. The developed proteomic method exploiting urinary exosomes might be of great value in search for diagnostic or prognostic biomarkers of especially metabolic and immune-related diseases. PMID:27805059

  6. Proteomic Analysis of Protein-Protein Interactions within the CSD Fe-S Cluster Biogenesis System

    PubMed Central

    Bolstad, Heather M.; Botelho, Danielle J.; Wood, Matthew J.

    2010-01-01

    Fe-S cluster biogenesis is of interest to many fields, including bioenergetics and gene regulation. The CSD system is one of three Fe-S cluster biogenesis systems in E. coli and is comprised of the cysteine desulfurase CsdA, the sulfur acceptor protein CsdE, and the E1-like protein CsdL. The biological role, biochemical mechanism, and protein targets of the system remain uncharacterized. Here we present that the active site CsdE C61 has a lowered pKa value of 6.5, which is nearly identical to that of C51 in the homologous SufE protein and which is likely critical for its function. We observed that CsdE forms disulfide bonds with multiple proteins and identified the proteins that copurify with CsdE. The identification of Fe-S proteins and both putative and established Fe-S cluster assembly (ErpA, glutaredoxin-3, glutaredoxin-4) and sulfur trafficking (CsdL, YchN) proteins supports the two-pathway model, in which the CSD system is hypothesized to synthesize both Fe-S clusters and other sulfur-containing cofactors. We suggest that the identified Fe-S cluster assembly proteins may be the scaffold and/or shuttle proteins for the CSD system. By comparison with previous analysis of SufE, we demonstrate that there is some overlap in the CsdE and SufE interactomes. PMID:20734996

  7. Protein array based interactome analysis of amyloid-β indicates an inhibition of protein translation.

    PubMed

    Virok, Dezso P; Simon, Dóra; Bozsó, Zsolt; Rajkó, Róbert; Datki, Zsolt; Bálint, Éva; Szegedi, Viktor; Janáky, Tamás; Penke, Botond; Fülöp, Lívia

    2011-04-01

    Oligomeric amyloid-β is currently of interest in amyloid-β mediated toxicity and the pathogenesis of Alzheimer's disease. Mapping the amyloid-β interaction partners could help to discover novel pathways in disease pathogenesis. To discover the amyloid-β interaction partners, we applied a protein array with more than 8100 unique recombinantly expressed human proteins. We identified 324 proteins as potential interactors of oligomeric amyloid-β. The Gene Ontology functional analysis of these proteins showed that oligomeric amyloid-β bound to multiple proteins with diverse functions both from extra and intracellular localizations. This undiscriminating binding phenotype indicates that multiple protein interactions mediate the toxicity of the oligomeric amyloid-β. The most highly impacted cellular system was the protein translation machinery. Oligomeric amyloid-β could bind to altogether 24 proteins involved in translation initiation and elongation. The binding of amyloid-β to purified rat hippocampal ribosomes validated the protein array results. More importantly, in vitro translation assays showed that the oligomeric amyloid-β had a concentration dependent inhibitory activity on translation. Our results indicate that the inhibited protein synthesis is one of the pathways that can be involved in the amyloid-beta induced neurotoxicity.

  8. Inferring Domain-Domain Interactions from Protein-Protein Interactions with Formal Concept Analysis

    PubMed Central

    Khor, Susan

    2014-01-01

    Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains. PMID:24586450

  9. Raman optical activity: a tool for protein structure analysis.

    PubMed

    Zhu, Fujiang; Isaacs, Neil W; Hecht, Lutz; Barron, Laurence D

    2005-10-01

    On account of its sensitivity to chirality, Raman optical activity (ROA), measured here as the intensity of a small, circularly polarized component in the scattered light using unpolarized incident light, is a powerful probe of protein structure and behavior. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration, and side chain conformations, and on structural elements present in unfolded states. This article describes the ROA technique and presents ROA spectra, recorded with a commercial instrument of novel design, of a selection of proteins to demonstrate how ROA may be used to readily distinguish between the main classes of protein structure. A principal component analysis illustrates how the many structure-sensitive bands in protein ROA spectra are favorable for applying pattern recognition techniques to determine structural relationships between different proteins.

  10. Synchrotron IR microspectroscopy for protein structure analysis: Potential and questions

    DOE PAGES

    Yu, Peiqiang

    2006-01-01

    Synchrotron radiation-based Fourier transform infrared microspectroscopy (S-FTIR) has been developed as a rapid, direct, non-destructive, bioanalytical technique. This technique takes advantage of synchrotron light brightness and small effective source size and is capable of exploring the molecular chemical make-up within microstructures of a biological tissue without destruction of inherent structures at ultra-spatial resolutions within cellular dimension. To date there has been very little application of this advanced technique to the study of pure protein inherent structure at a cellular level in biological tissues. In this review, a novel approach was introduced to show the potential of the newly developed, advancedmore » synchrotron-based analytical technology, which can be used to localize relatively “pure“ protein in the plant tissues and relatively reveal protein inherent structure and protein molecular chemical make-up within intact tissue at cellular and subcellular levels. Several complex protein IR spectra data analytical techniques (Gaussian and Lorentzian multi-component peak modeling, univariate and multivariate analysis, principal component analysis (PCA), and hierarchical cluster analysis (CLA) are employed to relatively reveal features of protein inherent structure and distinguish protein inherent structure differences between varieties/species and treatments in plant tissues. By using a multi-peak modeling procedure, RELATIVE estimates (but not EXACT determinations) for protein secondary structure analysis can be made for comparison purpose. The issues of pro- and anti-multi-peaking modeling/fitting procedure for relative estimation of protein structure were discussed. By using the PCA and CLA analyses, the plant molecular structure can be qualitatively separate one group from another, statistically, even though the spectral assignments are not known. The synchrotron-based technology provides a new approach for protein structure research in

  11. Atom depth analysis delineates mechanisms of protein intermolecular interactions

    SciTech Connect

    Alocci, Davide; Bernini, Andrea; Niccolai, Neri

    2013-07-12

    Highlights: •3D atom depth analysis is proposed to identify different layers in protein structures. •Amino acid contents for each layers have been analyzed for a large protein dataset. •Charged amino acids in the most external layer are present at very different extents. •Atom depth indexes of K residues reflect their side chains flexibility. •Mobile surface charges can be responsible for long range protein–protein recognition. -- Abstract: The systematic analysis of amino acid distribution, performed inside a large set of resolved protein structures, sheds light on possible mechanisms driving non random protein–protein approaches. Protein Data Bank entries have been selected using as filters a series of restrictions ensuring that the shape of protein surface is not modified by interactions with large or small ligands. 3D atom depth has been evaluated for all the atoms of the 2,410 selected structures. The amino acid relative population in each of the structural layers formed by grouping atoms on the basis of their calculated depths, has been evaluated. We have identified seven structural layers, the inner ones reproducing the core of proteins and the outer one incorporating their most protruding moieties. Quantitative analysis of amino acid contents of structural layers identified, as expected, different behaviors. Atoms of Q, R, K, N, D residues are increasingly more abundant in going from core to surfaces. An opposite trend is observed for V, I, L, A, C, and G. An intermediate behavior is exhibited by P, S, T, M, W, H, F and Y. The outer structural layer hosts predominantly E and K residues whose charged moieties, protruding from outer regions of the protein surface, reorient free from steric hindrances, determining specific electrodynamics maps. This feature may represent a protein signature for long distance effects, driving the formation of encounter complexes and the eventual short distance approaches that are required for protein–protein

  12. Genomewide analysis of phytochrome proteins in the phylum Basidiomycota.

    PubMed

    Lavín, José L; Ramírez, Lucía; Pisabarro, Antonio G; Oguiza, José A

    2015-09-01

    Phytochromes are photoreceptor proteins involved in the detection of the red and far-red regions of the visible light spectrum. Fungal phytochromes are hybrid histidine kinases with a conserved domain architecture composed of an N-terminal photosensory module and a C-terminal regulatory output module that includes the histidine kinase and response regulator receiver domains. In this study, we have analyzed the distribution, domain architecture, and phylogenetic analysis of phytochrome proteins in 47 published genome sequences among the phylum Basidiomycota. Genome analysis revealed that almost every genome of basidiomycetes contained at least one gene encoding a phytochrome protein. Domain architecture of fungal phytochromes was completely conserved in the identified phytochromes of basidiomycetes, and phylogenetic analysis clustered these proteins into clades related with the phylogenetic classification of this fungal phylum.

  13. Tetra Detector Analysis of Membrane Proteins

    PubMed Central

    Robbins, Rebecca A.; Stroud, Robert M.

    2014-01-01

    Well-characterized membrane protein detergent complexes (PDC) that are pure, homogenous and stable with minimized excess detergent micelles are essential for functional assays and crystallization studies. Procedural steps to measure the mass, size, shape, homogeneity and molecular composition of PDCs and their host detergent micelle using size exclusion chromatography (SEC) with a Viscotek tetra detector array (TDA; absorbance, refractive index, light scattering and viscosity detectors) are presented. The value of starting with a quality PDC sample, the precision and accuracy of the results, and the use of a digital bench top refractometer are emphasized. An alternate and simplified purification and characterization approach using SEC with dual absorbance and refractive index detectors to optimize detergent and lipid concentration while measuring the PDC homogeneity are also described. Applications relative to purification and characterization goals are illustrated as well. PMID:25081744

  14. Analysis of substructural variation in families of enzymatic proteins with applications to protein function prediction

    PubMed Central

    2010-01-01

    Background Structural variations caused by a wide range of physico-chemical and biological sources directly influence the function of a protein. For enzymatic proteins, the structure and chemistry of the catalytic binding site residues can be loosely defined as a substructure of the protein. Comparative analysis of drug-receptor substructures across and within species has been used for lead evaluation. Substructure-level similarity between the binding sites of functionally similar proteins has also been used to identify instances of convergent evolution among proteins. In functionally homologous protein families, shared chemistry and geometry at catalytic sites provide a common, local point of comparison among proteins that may differ significantly at the sequence, fold, or domain topology levels. Results This paper describes two key results that can be used separately or in combination for protein function analysis. The Family-wise Analysis of SubStructural Templates (FASST) method uses all-against-all substructure comparison to determine Substructural Clusters (SCs). SCs characterize the binding site substructural variation within a protein family. In this paper we focus on examples of automatically determined SCs that can be linked to phylogenetic distance between family members, segregation by conformation, and organization by homology among convergent protein lineages. The Motif Ensemble Statistical Hypothesis (MESH) framework constructs a representative motif for each protein cluster among the SCs determined by FASST to build motif ensembles that are shown through a series of function prediction experiments to improve the function prediction power of existing motifs. Conclusions FASST contributes a critical feedback and assessment step to existing binding site substructure identification methods and can be used for the thorough investigation of structure-function relationships. The application of MESH allows for an automated, statistically rigorous procedure

  15. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.

    PubMed

    Banks, Charles A S; Kong, Stephanie E; Washburn, Michael P

    2012-12-01

    Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

    PubMed

    Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

    2016-10-20

    RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.

  17. Protein Co-Expression Network Analysis (ProCoNA)

    SciTech Connect

    Gibbs, David L.; Baratt, Arie; Baric, Ralph; Kawaoka, Yoshihiro; Smith, Richard D.; Orwoll, Eric S.; Katze, Michael G.; Mcweeney, Shannon K.

    2013-06-01

    Biological networks are important for elucidating disease etiology due to their ability to model complex high dimensional data and biological systems. Proteomics provides a critical data source for such models, but currently lacks robust de novo methods for network construction, which could bring important insights in systems biology. We have evaluated the construction of network models using methods derived from weighted gene co-expression network analysis (WGCNA). We show that approximately scale-free peptide networks, composed of statistically significant modules, are feasible and biologically meaningful using two mouse lung experiments and one human plasma experiment. Within each network, peptides derived from the same protein are shown to have a statistically higher topological overlap and concordance in abundance, which is potentially important for inferring protein abundance. The module representatives, called eigenpeptides, correlate significantly with biological phenotypes. Furthermore, within modules, we find significant enrichment for biological function and known interactions (gene ontology and protein-protein interactions). Biological networks are important tools in the analysis of complex systems. In this paper we evaluate the application of weighted co-expression network analysis to quantitative proteomics data. Protein co-expression networks allow novel approaches for biological interpretation, quality control, inference of protein abundance, a framework for potentially resolving degenerate peptide-protein mappings, and a biomarker signature discovery.

  18. Computer analysis and structure prediction of nucleic acids and proteins.

    PubMed Central

    Kanehisa, M; Klein, P; Greif, P; DeLisi, C

    1984-01-01

    We have developed an integrated computer system for analysis of nucleic acid and protein sequences, which consists of sequence and structure databases, a relational database, and software for structural analysis. The system is potentially applicable to a number of problems in structural biology including predictive classification of the function and location of oncogene products. PMID:6546426

  19. Proteomic analysis of Toxocara canis excretory and secretory (TES) proteins.

    PubMed

    Sperotto, Rita Leal; Kremer, Frederico Schmitt; Aires Berne, Maria Elisabeth; Costa de Avila, Luciana F; da Silva Pinto, Luciano; Monteiro, Karina Mariante; Caumo, Karin Silva; Ferreira, Henrique Bunselmeyer; Berne, Natália; Borsuk, Sibele

    2017-01-01

    Toxocariasis is a neglected disease, and its main etiological agent is the nematode Toxocara canis. Serological diagnosis is performed by an enzyme-linked immunosorbent assay using T. canis excretory and secretory (TES) antigens produced by in vitro cultivation of larvae. Identification of TES proteins can be useful for the development of new diagnostic strategies since few TES components have been described so far. Herein, we report the results obtained by proteomic analysis of TES proteins using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. TES fractions were separated by one-dimensional SDS-PAGE and analyzed by LC-MS/MS. The MS/MS spectra were compared with a database of protein sequences deduced from the genome sequence of T. canis, and a total of 19 proteins were identified. Classification according to the signal peptide prediction using the SignalP server showed that seven of the identified proteins were extracellular, 10 had cytoplasmic or nuclear localization, while the subcellular localization of two proteins was unknown. Analysis of molecular functions by BLAST2GO showed that the majority of the gene ontology (GO) terms associated with the proteins present in the TES sample were associated with binding functions, including but not limited to protein binding (GO:0005515), inorganic ion binding (GO:0043167), and organic cyclic compound binding (GO:0097159). This study provides additional information about the exoproteome of T. canis, which can lead to the development of new strategies for diagnostics or vaccination.

  20. Analysis of protein composition and protein expression in the tear fluid of patients with congenital aniridia.

    PubMed

    Ihnatko, Robert; Edén, Ulla; Lagali, Neil; Dellby, Anette; Fagerholm, Per

    2013-12-06

    Aniridia is a rare congenital genetic disorder caused by haploinsuffiency of the PAX6 gene, the master gene for development of the eye. The expression of tear proteins in aniridia is unknown. To screen for proteins involved in the aniridia pathophysiology, the tear fluid of patients with diagnosed congenital aniridia was examined using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two-dimensional map of tear proteins in aniridia has been established and 7 proteins were differentially expressed with P<0.01 between aniridia patients and control subjects. Five of them were more abundant in healthy subjects, particularly α-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1 and two other proteins, zinc-α2-glycoprotein and lactoferrin were more expressed in the tears of aniridia patients. Moreover, immunoblot analysis revealed elevated levels of vascular endothelial growth factor (VEGF) in aniridia tears which is in concordance with clinical finding of pathological blood and lymph vessels in the central and peripheral cornea of aniridia patients. The proteins with different expression in patients' tears may be new candidate molecules involved in the pathophysiology of aniridia and thus may be helpful for development of novel treatment strategies for the symptomatic therapy of this vision threatening condition. This study is first to demonstrate protein composition and protein expression in aniridic tears and identifies proteins with different abundance in tear fluid from patients with congenital aniridia vs. healthy tears. © 2013 Elsevier B.V. All rights reserved.

  1. Proteomic analysis of the herpes simplex virus 1 virion protein 16 transactivator protein in infected cells.

    PubMed

    Suk, Hyung; Knipe, David M

    2015-06-01

    The herpes simplex virus 1 virion protein 16 (VP16) tegument protein forms a transactivation complex with the cellular proteins host cell factor 1 (HCF-1) and octamer-binding transcription factor 1 (Oct-1) upon entry into the host cell. VP16 has also been shown to interact with a number of virion tegument proteins and viral glycoprotein H to promote viral assembly, but no comprehensive study of the VP16 proteome has been performed at early times postinfection. We therefore performed a proteomic analysis of VP16-interacting proteins at 3 h postinfection. We confirmed the interaction of VP16 with HCF-1 and a large number of cellular Mediator complex proteins, but most surprisingly, we found that the major viral protein associating with VP16 is the infected cell protein 4 (ICP4) immediate-early (IE) transactivator protein. These results raise the potential for a new function for VP16 in associating with the IE ICP4 and playing a role in transactivation of early and late gene expression, in addition to its well-documented function in transactivation of IE gene expression.

  2. Protein complex prediction via dense subgraphs and false positive analysis.

    PubMed

    Hernandez, Cecilia; Mella, Carlos; Navarro, Gonzalo; Olivera-Nappa, Alvaro; Araya, Jaime

    2017-01-01

    Many proteins work together with others in groups called complexes in order to achieve a specific function. Discovering protein complexes is important for understanding biological processes and predict protein functions in living organisms. Large-scale and throughput techniques have made possible to compile protein-protein interaction networks (PPI networks), which have been used in several computational approaches for detecting protein complexes. Those predictions might guide future biologic experimental research. Some approaches are topology-based, where highly connected proteins are predicted to be complexes; some propose different clustering algorithms using partitioning, overlaps among clusters for networks modeled with unweighted or weighted graphs; and others use density of clusters and information based on protein functionality. However, some schemes still require much processing time or the quality of their results can be improved. Furthermore, most of the results obtained with computational tools are not accompanied by an analysis of false positives. We propose an effective and efficient mining algorithm for discovering highly connected subgraphs, which is our base for defining protein complexes. Our representation is based on transforming the PPI network into a directed acyclic graph that reduces the number of represented edges and the search space for discovering subgraphs. Our approach considers weighted and unweighted PPI networks. We compare our best alternative using PPI networks from Saccharomyces cerevisiae (yeast) and Homo sapiens (human) with state-of-the-art approaches in terms of clustering, biological metrics and execution times, as well as three gold standards for yeast and two for human. Furthermore, we analyze false positive predicted complexes searching the PDBe (Protein Data Bank in Europe) database in order to identify matching protein complexes that have been purified and structurally characterized. Our analysis shows that more than 50

  3. Linguistic feature analysis for protein interaction extraction

    PubMed Central

    2009-01-01

    Background The rapid growth of the amount of publicly available reports on biomedical experimental results has recently caused a boost of text mining approaches for protein interaction extraction. Most approaches rely implicitly or explicitly on linguistic, i.e., lexical and syntactic, data extracted from text. However, only few attempts have been made to evaluate the contribution of the different feature types. In this work, we contribute to this evaluation by studying the relative importance of deep syntactic features, i.e., grammatical relations, shallow syntactic features (part-of-speech information) and lexical features. For this purpose, we use a recently proposed approach that uses support vector machines with structured kernels. Results Our results reveal that the contribution of the different feature types varies for the different data sets on which the experiments were conducted. The smaller the training corpus compared to the test data, the more important the role of grammatical relations becomes. Moreover, deep syntactic information based classifiers prove to be more robust on heterogeneous texts where no or only limited common vocabulary is shared. Conclusion Our findings suggest that grammatical relations play an important role in the interaction extraction task. Moreover, the net advantage of adding lexical and shallow syntactic features is small related to the number of added features. This implies that efficient classifiers can be built by using only a small fraction of the features that are typically being used in recent approaches. PMID:19909518

  4. Biomimetic Coacervate Environments for Protein Analysis

    NASA Astrophysics Data System (ADS)

    Perry, Sarah; McCall, Patrick; Srivastava, Samavayan; Kovar, David; Gardel, Margaret; Tirrell, Matthew

    2015-03-01

    Living cells have evolved sophisticated intracellular organization strategies that are challenging to reproduce synthetically. Biomolecular function depends on both the structure of the molecule itself and the properties of the surrounding medium. The ability to simulate the in vivo environment and isolate biological networks for study in an artificial milieu without sacrificing the crowding, structure, and compartmentalization of a cellular environment, represent engineering challenges with tremendous potential to impact both biological studies and biomedical applications. Emerging experience has shown that polypeptide-based complex coacervation (electrostatically-driven liquid-liquid phase separation) produces a biomimetic microenvironment capable of tuning protein biochemical activity. We have investigated the effect of polypeptide-based coacervates on the dynamic self-assembly of cytoskeletal actin filaments. Coacervate materials are able to directly affect the nucleation and assembly dynamics. We observe effects that can be attributed to the length and chemical specificity of the encapsulating polypeptides, as well as the overall crowded nature of a polymer-rich coacervate phase. Coacervate-based systems are particularly attractive for use in biochemical assays because the compartmentalization afforded by liquid-liquid phase separation does not necessarily inhibit the transport of molecules across the compartmental barrier.

  5. A comparative protein function analysis databaseof different Leishmania strains

    PubMed Central

    Dikhit, Manas Ranjan; Nathasharma, Yangya Prasad; Patel, Lelin; Rana, Sindhu Prava; Sahoo, Ganesh Chandra; Das, Pradeep

    2011-01-01

    A complete understanding of different protein functional families and template information opens new avenues for novel drug development. Protein identification and analysis software performs a central role in the investigation of proteins and leads to the development of refined database for description of proteins of different Leishmania strains. There are certain databases for different strains that lack template information and functional family annotation. Rajendra Memorial Research Institute of Medical Sciences (RMRIMS) has developed a web-based unique database to provide information about functional families of different proteins and its template information in different Leishmania species. Based on the template information users can model the tertiary structure of protein. The database facilitates significant relationship between template information and possible protein functional families assigned to different proteins by SVMProt. This database is designed to provide comprehensive descriptions of certain important proteins found in four different species of Leishmania i.e. L. donovani, L. infantum, L. major and L. braziliensis. A specific characterization information table provides information related to species and specific functional families. This database aims to be a resource for scientists working on proteomics. The database is freely available at http://biomedinformri.org/calp/. PMID:21464840

  6. High-Bandwidth Protein Analysis Using Solid-State Nanopores

    PubMed Central

    Larkin, Joseph; Henley, Robert Y.; Muthukumar, Murugappan; Rosenstein, Jacob K.; Wanunu, Meni

    2014-01-01

    High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D0) by a factor of ∼50, and are voltage-independent in the regime tested. We reason that deviations of D from D0 are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved. PMID:24507610

  7. CISAPS: Complex Informational Spectrum for the Analysis of Protein Sequences.

    PubMed

    Chrysostomou, Charalambos; Seker, Huseyin; Aydin, Nizamettin

    2015-01-01

    Complex informational spectrum analysis for protein sequences (CISAPS) and its web-based server are developed and presented. As recent studies show, only the use of the absolute spectrum in the analysis of protein sequences using the informational spectrum analysis is proven to be insufficient. Therefore, CISAPS is developed to consider and provide results in three forms including absolute, real, and imaginary spectrum. Biologically related features to the analysis of influenza A subtypes as presented as a case study in this study can also appear individually either in the real or imaginary spectrum. As the results presented, protein classes can present similarities or differences according to the features extracted from CISAPS web server. These associations are probable to be related with the protein feature that the specific amino acid index represents. In addition, various technical issues such as zero-padding and windowing that may affect the analysis are also addressed. CISAPS uses an expanded list of 611 unique amino acid indices where each one represents a different property to perform the analysis. This web-based server enables researchers with little knowledge of signal processing methods to apply and include complex informational spectrum analysis to their work.

  8. CISAPS: Complex Informational Spectrum for the Analysis of Protein Sequences

    PubMed Central

    Seker, Huseyin; Aydin, Nizamettin

    2015-01-01

    Complex informational spectrum analysis for protein sequences (CISAPS) and its web-based server are developed and presented. As recent studies show, only the use of the absolute spectrum in the analysis of protein sequences using the informational spectrum analysis is proven to be insufficient. Therefore, CISAPS is developed to consider and provide results in three forms including absolute, real, and imaginary spectrum. Biologically related features to the analysis of influenza A subtypes as presented as a case study in this study can also appear individually either in the real or imaginary spectrum. As the results presented, protein classes can present similarities or differences according to the features extracted from CISAPS web server. These associations are probable to be related with the protein feature that the specific amino acid index represents. In addition, various technical issues such as zero-padding and windowing that may affect the analysis are also addressed. CISAPS uses an expanded list of 611 unique amino acid indices where each one represents a different property to perform the analysis. This web-based server enables researchers with little knowledge of signal processing methods to apply and include complex informational spectrum analysis to their work. PMID:25632276

  9. Proteomic analysis of protein adsorption capacity of different haemodialysis membranes.

    PubMed

    Urbani, Andrea; Lupisella, Santina; Sirolli, Vittorio; Bucci, Sonia; Amoroso, Luigi; Pavone, Barbara; Pieroni, Luisa; Sacchetta, Paolo; Bonomini, Mario

    2012-04-01

    Protein-adsorptive properties are a key feature of membranes used for haemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with chemical feature of the membrane surfaces. Dialyzers composed of either cellulose triacetate (Sureflux 50 L, effective surface area 0.5 m(2); Nipro Corporation, Japan) or the polysulfone-based helixone (FX40, effective surface area 0.4 m(2); Fresenius Medical Care AG, Germany) materials were employed to develop an ex vivo apparatus to study protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by a proteomic approach. The profiling strategy was based on 2D-electrophoresis separation of desorbed protein coupled to MALDI-TOF/TOF analysis. The total protein adsorption was not significantly different between the two materials. An average of 179 protein spots was visualised for helixone membranes while a map of retained proteins of cellulose triacetate membranes was made up of 239 protein spots. The cellulose triacetate material showed a higher binding capacity for albumin and apolipoprotein. In fact, a number of different protein spots belonging to the gene transcript of albumin were visible in the cellulose triacetate map. In contrast, helixone bound only a small proportion of albumin, while proved to be particularly active in retaining protein associated with the coagulation cascade, such as the fibrinogen isoforms. Our data indicate that proteomic techniques are a useful approach for the investigation of proteins surface-adsorbed onto haemodialysis membranes, and may provide a molecular base for the interpretation of the efficacy and safety of anticoagulation treatment during renal replacement therapy.

  10. Identification Of Protein Vaccine Candidates Using Comprehensive Proteomic Analysis Strategies

    DTIC Science & Technology

    2007-12-01

    133. Jefferies, J. R., A. M. Campbell, A. J. van Rossum, et al. 2001. Proteomic analysis of Fasciola hepatica excretory-secretory products...performed on other organisms prevalent in human disease, such as the analysis of excreted proteins from the human parasitic liver fluke Fasciola ... hepatica , in the search for potential vaccine candidates. 133 More recent studies have employed MudPIT analysis for the identification of potential

  11. Analysis of membrane proteins in metagenomics: networks of correlated environmental features and protein families.

    PubMed

    Patel, Prianka V; Gianoulis, Tara A; Bjornson, Robert D; Yip, Kevin Y; Engelman, Donald M; Gerstein, Mark B

    2010-07-01

    Recent metagenomics studies have begun to sample the genomic diversity among disparate habitats and relate this variation to features of the environment. Membrane proteins are an intuitive, but thus far overlooked, choice in this type of analysis as they directly interact with the environment, receiving signals from the outside and transporting nutrients. Using global ocean sampling (GOS) data, we found nearly approximately 900,000 membrane proteins in large-scale metagenomic sequence, approximately a fifth of which are completely novel, suggesting a large space of hitherto unexplored protein diversity. Using GPS coordinates for the GOS sites, we extracted additional environmental features via interpolation from the World Ocean Database, the National Center for Ecological Analysis and Synthesis, and empirical models of dust occurrence. This allowed us to study membrane protein variation in terms of natural features, such as phosphate and nitrate concentrations, and also in terms of human impacts, such as pollution and climate change. We show that there is widespread variation in membrane protein content across marine sites, which is correlated with changes in both oceanographic variables and human factors. Furthermore, using these data, we developed an approach, protein families and environment features network (PEN), to quantify and visualize the correlations. PEN identifies small groups of covarying environmental features and membrane protein families, which we call "bimodules." Using this approach, we find that the affinity of phosphate transporters is related to the concentration of phosphate and that the occurrence of iron transporters is connected to the amount of shipping, pollution, and iron-containing dust.

  12. Analysis of NCL Proteins from an Evolutionary Standpoint

    PubMed Central

    Muzaffar, Neda E; Pearce, David A

    2008-01-01

    The Neuronal Ceroid Lipofuscinoses (NCLs) are the most common group of neurodegenerative disorders of childhood. While mutations in eight different genes have been shown to be responsible for these clinically distinct types of NCL, the NCLs share many clinical and pathological similarities. We have conducted an exhaustive Basic Local Alignment Search Tool (BLAST) analysis of the human protein sequences for each of the eight known NCL proteins- CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8 and CLN10. The number of homologous species per CLN-protein identified by BLAST searches varies depending on the parameters set for the BLAST search. For example, a lower threshold is able to pull up more homologous sequences whereas a higher threshold decreases this number. Nevertheless, the clade confines are consistent despite this variation in BLAST searching parameters. Further phylogenetic analyses on the appearance of NCL proteins through evolution reveals a different time line for the appearance of the CLN-proteins. Moreover, divergence of each protein shows a different pattern, providing important clues on the evolving role of these proteins. We present and review in-depth bioinformatic analysis of the NCL proteins and classify the CLN-proteins into families based on their structures and evolutionary relationships, respectively. Based on these analyses, we have grouped the CLN-proteins into common clades indicating a common evolving pathway within the evolutionary tree of life. CLN2 is grouped in Eubacteria, CLN1 and CLN10 in Viridiplantae, CLN3 in Fungi/ Metazoa, CLN7 in Bilateria and CLN5, CLN6 and CLN8 in Euteleostomi. PMID:19440452

  13. MIPS: analysis and annotation of proteins from whole genomes.

    PubMed

    Mewes, H W; Amid, C; Arnold, R; Frishman, D; Güldener, U; Mannhaupt, G; Münsterkötter, M; Pagel, P; Strack, N; Stümpflen, V; Warfsmann, J; Ruepp, A

    2004-01-01

    The Munich Information Center for Protein Sequences (MIPS-GSF), Neuherberg, Germany, provides protein sequence-related information based on whole-genome analysis. The main focus of the work is directed toward the systematic organization of sequence-related attributes as gathered by a variety of algorithms, primary information from experimental data together with information compiled from the scientific literature. MIPS maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the database of complete cDNAs (German Human Genome Project, NGFN), the database of mammalian protein-protein interactions (MPPI), the database of FASTA homologies (SIMAP), and the interface for the fast retrieval of protein-associated information (QUIPOS). The Arabidopsis thaliana database, the rice database, the plant EST databases (MATDB, MOsDB, SPUTNIK), as well as the databases for the comprehensive set of genomes (PEDANT genomes) are described elsewhere in the 2003 and 2004 NAR database issues, respectively. All databases described, and the detailed descriptions of our projects can be accessed through the MIPS web server (http://mips.gsf.de).

  14. A novel approach for oxidation analysis of therapeutic proteins.

    PubMed

    Turyan, Iva; Khatwani, Nikhil; Sosic, Zoran; Jayawickreme, Shiranthi; Mandler, Daniel

    2016-02-01

    Measuring and monitoring of protein oxidation modifications is important for biopharmaceutical process development and stability assessment during long-term storage. Currently available methods for biomolecules oxidation analysis use time-consuming peptide mapping analysis. Therefore, it is desirable to develop high-throughput methods for advanced process control of protein oxidation. Here, we present a novel approach by which oxidative protein modifications are monitored by an indirect potentiometric method. The method is based on adding an electron mediator, which enhances electron transfer (ET) between all redox species and the electrode surface. Specifically, the procedure involves measuring the sharp change in the open circuit potential (OCP) for the mediator system (redox couple) as a result of its interaction with the oxidized protein species in the solution. Application of Pt and Ag/AgCl microelectrodes allowed for a high-sensitivity protein oxidation analysis. We found that the Ru(NH3)6(2+/3+) redox couple is suitable for measuring the total oxidation of a wide range of therapeutic proteins between 1.1 and 13.6%. Accuracy determined by comparing with the known percentage oxidation of the reference standard showed that percentage oxidation determined for each sample was within ± 20% of the expected percentage oxidation determined by mass spectrometry.

  15. Clustering and Network Analysis of Reverse Phase Protein Array Data.

    PubMed

    Byron, Adam

    2017-01-01

    Molecular profiling of proteins and phosphoproteins using a reverse phase protein array (RPPA) platform, with a panel of target-specific antibodies, enables the parallel, quantitative proteomic analysis of many biological samples in a microarray format. Hence, RPPA analysis can generate a high volume of multidimensional data that must be effectively interrogated and interpreted. A range of computational techniques for data mining can be applied to detect and explore data structure and to form functional predictions from large datasets. Here, two approaches for the computational analysis of RPPA data are detailed: the identification of similar patterns of protein expression by hierarchical cluster analysis and the modeling of protein interactions and signaling relationships by network analysis. The protocols use freely available, cross-platform software, are easy to implement, and do not require any programming expertise. Serving as data-driven starting points for further in-depth analysis, validation, and biological experimentation, these and related bioinformatic approaches can accelerate the functional interpretation of RPPA data.

  16. Principal component analysis based methodology to distinguish protein SERS spectra

    NASA Astrophysics Data System (ADS)

    Das, G.; Gentile, F.; Coluccio, M. L.; Perri, A. M.; Nicastri, A.; Mecarini, F.; Cojoc, G.; Candeloro, P.; Liberale, C.; De Angelis, F.; Di Fabrizio, E.

    2011-05-01

    Surface-enhanced Raman scattering (SERS) substrates were fabricated using electro-plating and e-beam lithography techniques. Nano-structures were obtained comprising regular arrays of gold nanoaggregates with a diameter of 80 nm and a mutual distance between the aggregates (gap) ranging from 10 to 30 nm. The nanopatterned SERS substrate enabled to have better control and reproducibility on the generation of plasmon polaritons (PPs). SERS measurements were performed for various proteins, namely bovine serum albumin (BSA), myoglobin, ferritin, lysozyme, RNase-B, α-casein, α-lactalbumin and trypsin. Principal component analysis (PCA) was used to organize and classify the proteins on the basis of their secondary structure. Cluster analysis proved that the error committed in the classification was of about 14%. In the paper, it was clearly shown that the combined use of SERS measurements and PCA analysis is effective in categorizing the proteins on the basis of secondary structure.

  17. Proteomic analysis of amphiphilic proteins of hexaploid wheat kernels.

    PubMed

    Amiour, Nardjis; Merlino, Marielle; Leroy, Philippe; Branlard, Gérard

    2002-06-01

    Wheat proteins and specially gluten proteins have been well studied and are closely associated with baking products. Amphiphilic proteins (proteins that are soluble using nonionic detergent Triton X-114 ) also play an important role in wheat quality. Some of them, like puroindolines, are lipid binding proteins, and are strongly linked to dough foaming properties and to fine crumb texture. However many amphiphilic proteins are still unknown and both their physiological and technological functions remain to be analysed. In order to explore these proteins, proteomic analysis was carried out using 81 F9 lines, progeny obtained from an interspecific cross "W7984"x"Opata", and already used to built a map of more than 2000 molecular markers (International Triticeae Mapping Initiative, ITMImap). Two-dimensional electrophoresis (immobilized pH gradient (pH 6-11)x sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed on amphiphilic proteins with three to five replicates for each line. Silver stained gels were analysed using Melanie 3 software. Genetic determinism was carried out on 170 spots segregating between the two parental hexaploïd wheats. Many of these spots were mapped on different chromosomes of the ITMImap. Spots of interest were identified using matrix-assisted laser desorption/ionization-time of flight and some of them were partly sequenced using electrospray ionization-tandem mass spectrometry. This proteomic approach provided some very useful information about some proteic components linked to bread wheat quality and particularly to kernel hardness.

  18. Quantitative Proteomic Approaches for Analysis of Protein S-Nitrosylation.

    PubMed

    Qu, Zhe; Greenlief, C Michael; Gu, Zezong

    2016-01-04

    S-Nitrosylation is a redox-based post-translational modification of a protein in response to nitric oxide (NO) signaling, and it participates in a variety of processes in diverse biological systems. The significance of this type of protein modification in health and diseases is increasingly recognized. In the central nervous system, aberrant S-nitrosylation, due to excessive NO production, is known to cause protein misfolding, mitochondrial dysfunction, transcriptional dysregulation, and neuronal death. This leads to an altered physiological state and consequently contributes to pathogenesis of neurodegenerative disorders. To date, much effort has been made to understand the mechanisms underlying protein S-nitrosylation, and several approaches have been developed to unveil S-nitrosylated proteins from different organisms. Interest in determining the dynamic changes of protein S-nitrosylation under different physiological and pathophysiological conditions has underscored the need for the development of quantitative proteomic approaches. Currently, both gel-based and gel-free mass spectrometry-based quantitative methods are widely used, and they each have advantages and disadvantages but may also be used together to produce complementary data. This review evaluates current available quantitative proteomic techniques for the analysis of protein S-nitrosylation and highlights recent advances, with emphasis on applications in neurodegenerative diseases. An important goal is to provide a comprehensive guide of feasible quantitative proteomic methodologies for examining protein S-nitrosylation in research to yield insights into disease mechanisms, diagnostic biomarkers, and drug discovery.

  19. Global Analysis of Protein Expression of Inner Ear Hair Cells.

    PubMed

    Hickox, Ann E; Wong, Ann C Y; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel; Yates, John R; Ryan, Allen F; Savas, Jeffrey N

    2017-02-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

  20. Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry.

    PubMed

    Belov, Arseniy M; Viner, Rosa; Santos, Marcia R; Horn, David M; Bern, Marshall; Karger, Barry L; Ivanov, Alexander R

    2017-09-05

    Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). Graphical Abstract ᅟ.

  1. Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Belov, Arseniy M.; Viner, Rosa; Santos, Marcia R.; Horn, David M.; Bern, Marshall; Karger, Barry L.; Ivanov, Alexander R.

    2017-09-01

    Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). [Figure not available: see fulltext.

  2. Protein interaction discovery using parallel analysis of translated ORFs (PLATO)

    PubMed Central

    Gao, Geng; Somwar, Romel; Zhang, Zijuan; Laserson, Uri; Ciccia, Alberto; Pavlova, Natalya; Church, George; Zhang, Wei; Kesari, Santosh; Elledge, Stephen J.

    2014-01-01

    Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method for human proteins by identifying known and previously unidentified interacting partners of LYN kinase, patient autoantibodies, and the small-molecules gefitinib and dasatinib. PMID:23503679

  3. Genomic analysis of membrane protein families: abundance and conserved motifs

    PubMed Central

    Liu, Yang; Engelman, Donald M; Gerstein, Mark

    2002-01-01

    Background Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. Results Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. Conclusions We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families. PMID:12372142

  4. Construction and analysis of protein-protein interaction networks based on proteomics data of prostate cancer

    PubMed Central

    CHEN, CHEN; SHEN, HONG; ZHANG, LI-GUO; LIU, JIAN; CAO, XIAO-GE; YAO, AN-LIANG; KANG, SHAO-SAN; GAO, WEI-XING; HAN, HUI; CAO, FENG-HONG; LI, ZHI-GUO

    2016-01-01

    Currently, using human prostate cancer (PCa) tissue samples to conduct proteomics research has generated a large amount of data; however, only a very small amount has been thoroughly investigated. In this study, we manually carried out the mining of the full text of proteomics literature that involved comparisons between PCa and normal or benign tissue and identified 41 differentially expressed proteins verified or reported more than 2 times from different research studies. We regarded these proteins as seed proteins to construct a protein-protein interaction (PPI) network. The extended network included one giant network, which consisted of 1,264 nodes connected via 1,744 edges, and 3 small separate components. The backbone network was then constructed, which was derived from key nodes and the subnetwork consisting of the shortest path between seed proteins. Topological analyses of these networks were conducted to identify proteins essential for the genesis of PCa. Solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4) had the highest closeness centrality located in the center of each network, and the highest betweenness centrality and largest degree in the backbone network. Tubulin, beta 2C (TUBB2C) had the largest degree in the giant network and subnetwork. In addition, using module analysis of the whole PPI network, we obtained a densely connected region. Functional annotation indicated that the Ras protein signal transduction biological process, mitogen-activated protein kinase (MAPK), neurotrophin and the gonadotropin-releasing hormone (GnRH) signaling pathway may play an important role in the genesis and development of PCa. Further investigation of the SLC2A4, TUBB2C proteins, and these biological processes and pathways may therefore provide a potential target for the diagnosis and treatment of PCa. PMID:27121963

  5. Construction and analysis of protein-protein interaction networks based on proteomics data of prostate cancer.

    PubMed

    Chen, Chen; Shen, Hong; Zhang, Li-Guo; Liu, Jian; Cao, Xiao-Ge; Yao, An-Liang; Kang, Shao-San; Gao, Wei-Xing; Han, Hui; Cao, Feng-Hong; Li, Zhi-Guo

    2016-06-01

    Currently, using human prostate cancer (PCa) tissue samples to conduct proteomics research has generated a large amount of data; however, only a very small amount has been thoroughly investigated. In this study, we manually carried out the mining of the full text of proteomics literature that involved comparisons between PCa and normal or benign tissue and identified 41 differentially expressed proteins verified or reported more than 2 times from different research studies. We regarded these proteins as seed proteins to construct a protein-protein interaction (PPI) network. The extended network included one giant network, which consisted of 1,264 nodes connected via 1,744 edges, and 3 small separate components. The backbone network was then constructed, which was derived from key nodes and the subnetwork consisting of the shortest path between seed proteins. Topological analyses of these networks were conducted to identify proteins essential for the genesis of PCa. Solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4) had the highest closeness centrality located in the center of each network, and the highest betweenness centrality and largest degree in the backbone network. Tubulin, beta 2C (TUBB2C) had the largest degree in the giant network and subnetwork. In addition, using module analysis of the whole PPI network, we obtained a densely connected region. Functional annotation indicated that the Ras protein signal transduction biological process, mitogen-activated protein kinase (MAPK), neurotrophin and the gonadotropin-releasing hormone (GnRH) signaling pathway may play an important role in the genesis and development of PCa. Further investigation of the SLC2A4, TUBB2C proteins, and these biological processes and pathways may therefore provide a potential target for the diagnosis and treatment of PCa.

  6. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    PubMed Central

    Doktycz, M. J.; Qi, H.; Morrell-Falvey, J. L.

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction. Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors. PMID:23165043

  7. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    DOE PAGES

    Venkatraman, S.; Doktycz, M. J.; Qi, H.; ...

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

  8. Construction and analysis of the protein-protein interaction network related to essential hypertension

    PubMed Central

    2013-01-01

    Background Essential hypertension (EH) is a complex disease as a consequence of interaction between environmental factors and genetic background, but the pathogenesis of EH remains elusive. The emerging tools of network medicine offer a platform to explore a complex disease at system level. In this study, we aimed to identify the key proteins and the biological regulatory pathways involving in EH and further to explore the molecular connectivities between these pathways by the topological analysis of the Protein-protein interaction (PPI) network. Result The extended network including one giant network consisted of 535 nodes connected via 2572 edges and two separated small networks. 27 proteins with high BC and 28 proteins with large degree have been identified. NOS3 with highest BC and Closeness centrality located in the centre of the network. The backbone network derived from high BC proteins presents a clear and visual overview which shows all important regulatory pathways for blood pressure (BP) and the crosstalk between them. Finally, the robustness of NOS3 as central protein and accuracy of backbone were validated by 287 test networks. Conclusion Our finding suggests that blood pressure variation is orchestrated by an integrated PPI network centered on NOS3. PMID:23587307

  9. Analysis of Protein Glycosylation and Phosphorylation Using Liquid Phase Separation, Protein Microarray Technology, and Mass Spectrometry

    PubMed Central

    Zhao, Jia; Patwa, Tasneem H.; Pal, Manoj; Qiu, Weilian; Lubman, David M.

    2010-01-01

    Summary Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to the onset and progression of cancer and other disease states. In this protocol, strategies for monitoring the changes in protein glycosylation and phosphorylation in serum or tissue cells on a global scale and specifically characterizing these alterations are included. The technique is based on lectin affinity enrichment for glycoproteins, all liquid-phase two-dimensional fractionation, protein microarray, and mass spectrometry technology. Proteins are separated based on pI in the first dimension using chromatofocusing (CF) or liquid isoelectric focusing (IEF) followed by the second-dimension separation using nonporous silica RP-HPLC. Five lectins with different binding specificities to glycan structures are used for screening glycosylation patterns in human serum through a biotin–streptavidin system. Fluorescent phosphodyes and phosphospecific antibodies are employed to detect specific phosphorylated proteins in cell lines or human tissues. The purified proteins of interest are identified by peptide sequencing. Their modifications including glycosylation and phosphorylation could be further characterized by mass-spectrometry-based approaches. These strategies can be used in biological samples for large-scale glycoproteome/phosphoproteome screening as well as for individual protein modification analysis. PMID:19241043

  10. Analysis of protein glycosylation and phosphorylation using liquid phase separation, protein microarray technology, and mass spectrometry.

    PubMed

    Zhao, Jia; Patwa, Tasneem H; Pal, Manoj; Qiu, Weilian; Lubman, David M

    2009-01-01

    Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to the onset and progression of cancer and other disease states. In this protocol, strategies for monitoring the changes in protein glycosylation and phosphorylation in serum or tissue cells on a global scale and specifically characterizing these alterations are included. The technique is based on lectin affinity enrichment for glycoproteins, all liquid-phase two-dimensional fractionation, protein microarray, and mass spectrometry technology. Proteins are separated based on pI in the first dimension using chromatofocusing (CF) or liquid isoelectric focusing (IEF) followed by the second-dimension separation using nonporous silica RP-HPLC. Five lectins with different binding specificities to glycan structures are used for screening glycosylation patterns in human serum through a biotin streptavidin system. Fluorescent phosphodyes and phosphospecific antibodies are employed to detect specific phosphorylated proteins in cell lines or human tissues. The purified proteins of interest are identified by peptide sequencing. Their modifications including glycosylation and phosphorylation could be further characterized by mass-spectrometry-based approaches. These strategies can be used in biological samples for large-scale glycoproteome/phosphoproteome screening as well as for individual protein modification analysis.

  11. Detailed Analysis of Protein Topology of Extracellular Vesicles–Evidence of Unconventional Membrane Protein Orientation

    PubMed Central

    Cvjetkovic, Aleksander; Jang, Su Chul; Konečná, Barbora; Höög, Johanna L.; Sihlbom, Carina; Lässer, Cecilia; Lötvall, Jan

    2016-01-01

    Extracellular vesicles (EVs) are important mediators of intercellular communication that change the recipient cell by shuttling lipids, RNA, or protein cargo between cells. Here, we investigate the topology of the protein cargo found in EVs, as this topology can fundamentally influence the biological effects of EVs. A multiple proteomics approach, combining proteinase treatment and biotin tagging, shows that many proteins of cytosolic origin are localized on the surface of EVs. A detailed analysis of the EV proteome at the peptide level revealed that a number of EV membrane proteins are present in a topologically reversed orientation compared to what is annotated. Two examples of such proteins, SCAMP3 and STX4, were confirmed to have a reversed topology. This reversed typology was determined using flow cytometry and fluorescent microscopy with antibodies directed toward their cytoplasmic epitopes. These results describe a novel workflow to define the EV proteome and the orientation of each protein, including membrane protein topology. These data are fundamentally important to understanding the EV proteome and required to fully explain EV biogenesis as well as biological function in recipient cells. PMID:27821849

  12. Immunocytochemistry and protein analysis suggest that reptilian claws contain small high cysteine-glycine proteins.

    PubMed

    Alibardi, L; Toni, M

    2009-06-01

    The present study analyzes the structure and the main proteins of reptilian claws. Mature claws are formed by two to four layers of keratinocytes, a transitional layer of spindle-shaped cells and a thick corneous layer. Transitional cells elongate and merge into a compact corneous layer that is immunoreactive for beta-keratins, now indicated as sauropsid keratin-associated proteins (sKAPs). Most proteins extracted from claws in representative reptiles have a molecular weight of 13-20kDa, an acidic to basic isoelectric point, and are identified from the positive immunoreactivity to beta-keratin antibodies. The comparative analysis between lizard and avian claw beta-keratins shows the presence of an internal region of 20 amino acids with the highest identity, indicated as core-box, within an extended 32-amino acid region with a prevalent beta-sheet secondary conformation. This region is structurally equivalent to a 32-amino acid region present in scale beta-keratins of most reptiles. Both reptilian and avian keratins contain glycine-rich regions for stabilization of the beta-keratin polymer. The N- and C-regions contain most cysteine for disulphide-bonds formation. Claw proteins contain higher amount of cysteine and glycine than other scale proteins, suggesting that claw proteins are specialized cysteine-glycine-rich proteins suited to produce a very hard corneous material.

  13. Kojak: Efficient analysis of chemically cross-linked protein complexes

    PubMed Central

    Hoopmann, Michael R.; Zelter, Alex; Johnson, Richard S.; Riffle, Michael; MacCoss, Michael J.; Davis, Trisha N.; Moritz, Robert L.

    2015-01-01

    Protein chemical cross-linking and mass spectrometry enable the analysis of protein-protein interactions and protein topologies, however complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein-protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms, and can identify cross-linked peptides using many different chemical cross-linkers, with or without heavy isotope labels. Kojak was used to analyze both novel and existing datasets, and was compared with existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms, and equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing datasets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods. PMID:25812159

  14. MIPS: analysis and annotation of proteins from whole genomes

    PubMed Central

    Mewes, H. W.; Amid, C.; Arnold, R.; Frishman, D.; Güldener, U.; Mannhaupt, G.; Münsterkötter, M.; Pagel, P.; Strack, N.; Stümpflen, V.; Warfsmann, J.; Ruepp, A.

    2004-01-01

    The Munich Information Center for Protein Sequences (MIPS-GSF), Neuherberg, Germany, provides protein sequence-related information based on whole-genome analysis. The main focus of the work is directed toward the systematic organization of sequence-related attributes as gathered by a variety of algorithms, primary information from experimental data together with information compiled from the scientific literature. MIPS maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the database of complete cDNAs (German Human Genome Project, NGFN), the database of mammalian protein–protein interactions (MPPI), the database of FASTA homologies (SIMAP), and the interface for the fast retrieval of protein-associated information (QUIPOS). The Arabidopsis thaliana database, the rice database, the plant EST databases (MATDB, MOsDB, SPUTNIK), as well as the databases for the comprehensive set of genomes (PEDANT genomes) are described elsewhere in the 2003 and 2004 NAR database issues, respectively. All databases described, and the detailed descriptions of our projects can be accessed through the MIPS web server (http://mips.gsf.de). PMID:14681354

  15. Use of morphological analysis in protein name recognition.

    PubMed

    Yamamoto, Kaoru; Kudo, Taku; Konagaya, Akihiko; Matsumoto, Yuji

    2004-12-01

    Protein name recognition aims to detect each and every protein names appearing in a PubMed abstract. The task is not simple, as the graphic word boundary (space separator) assumed in conventional preprocessing does not necessarily coincide with the protein name boundary. Such boundary disagreement caused by tokenization ambiguity has usually been ignored in conventional preprocessing of general English. In this paper, we argue that boundary disagreement poses serious limitations in biomedical English text processing, not to mention protein name recognition. Our key idea for dealing with the boundary disagreement is to apply techniques used in Japanese morphological analysis where there are no word boundaries. Having evaluated the proposed method with GENIA corpus 3.02, we obtain F-measure of 69.01 on a strict criterion and 79.32 on a relaxed criterion. The result is comparable to other published work in protein name recognition, without resorting to manually prepared ad hoc feature engineering. Further, compared to the conventional preprocessing, the use of morphological analysis as preprocessing improves the performance of protein name recognition and reduces the execution time.

  16. Modular arrangement of proteins as inferred from analysis of homology.

    PubMed Central

    Sonnhammer, E. L.; Kahn, D.

    1994-01-01

    The structure of many proteins consists of a combination of discrete modules that have been shuffled during evolution. Such modules can frequently be recognized from the analysis of homology. Here we present a systematic analysis of the modular organization of all sequenced proteins. To achieve this we have developed an automatic method to identify protein domains from sequence comparisons. Homologous domains can then be clustered into consistent families. The method was applied to all 21,098 nonfragment protein sequences in SWISS-PROT 21.0, which was automatically reorganized into a comprehensive protein domain database, ProDom. We have constructed multiple sequence alignments for each domain family in ProDom, from which consensus sequences were generated. These nonreduntant domain consensuses are useful for fast homology searches. Domain organization in ProDom is exemplified for proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PEP:PTS) and for bacterial 2-component regulators. We provide 2 examples of previously unrecognized domain arrangements discovered with the help of ProDom. PMID:8019419

  17. Insights from the structural analysis of protein heterodimer interfaces

    PubMed Central

    Sowmya, Gopichandran; Anita, Sathyanarayanan; Kangueane, Pandjassarame

    2011-01-01

    Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions. PMID:21572879

  18. Proteomic analysis of proteins involved in spermiogenesis in mouse.

    PubMed

    Guo, Xuejiang; Shen, Jian; Xia, Zhengrong; Zhang, Rui; Zhang, Ping; Zhao, Chun; Xing, Jun; Chen, Ling; Chen, Wen; Lin, Min; Huo, Ran; Su, Bing; Zhou, Zuomin; Sha, Jiahao

    2010-03-05

    Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

  19. Insights from the structural analysis of protein heterodimer interfaces.

    PubMed

    Sowmya, Gopichandran; Anita, Sathyanarayanan; Kangueane, Pandjassarame

    2011-05-07

    Protein heterodimer complexes are often involved in catalysis, regulation, assembly, immunity and inhibition. This involves the formation of stable interfaces between the interacting partners. Hence, it is of interest to describe heterodimer interfaces using known structural complexes. We use a non-redundant dataset of 192 heterodimer complex structures from the protein databank (PDB) to identify interface residues and describe their interfaces using amino-acids residue property preference. Analysis of the dataset shows that the heterodimer interfaces are often abundant in polar residues. The analysis also shows the presence of two classes of interfaces in heterodimer complexes. The first class of interfaces (class A) with more polar residues than core but less than surface is known. These interfaces are more hydrophobic than surfaces, where protein-protein binding is largely hydrophobic. The second class of interfaces (class B) with more polar residues than core and surface is shown. These interfaces are more polar than surfaces, where binding is mainly polar. Thus, these findings provide insights to the understanding of protein-protein interactions.

  20. Analysis of proteins and proteomes by mass spectrometry.

    PubMed

    Mann, M; Hendrickson, R C; Pandey, A

    2001-01-01

    A decade after the discovery of electrospray and matrix-assisted laser desorption ionization (MALDI), methods that finally allowed gentle ionization of large biomolecules, mass spectrometry has become a powerful tool in protein analysis and the key technology in the emerging field of proteomics. The success of mass spectrometry is driven both by innovative instrumentation designs, especially those operating on the time-of-flight or ion-trapping principles, and by large-scale biochemical strategies, which use mass spectrometry to detect the isolated proteins. Any human protein can now be identified directly from genome databases on the basis of minimal data derived by mass spectrometry. As has already happened in genomics, increased automation of sample handling, analysis, and the interpretation of results will generate an avalanche of qualitative and quantitative proteomic data. Protein-protein interactions can be analyzed directly by precipitation of a tagged bait followed by mass spectrometric identification of its binding partners. By these and similar strategies, entire protein complexes, signaling pathways, and whole organelles are being characterized. Posttranslational modifications remain difficult to analyze but are starting to yield to generic strategies.

  1. Cross-Pharmacology Analysis of G Protein-Coupled Receptors

    PubMed Central

    Briansó, Ferran; Carrascosa, Maria C.; Oprea, Tudor I.; Mestres, Jordi

    2013-01-01

    The degree of applicability of chemogenomic approaches to protein families depends on the accuracy and completeness of pharmacological data and the corresponding level of pharmacological similarity observed among their protein members. The recent public domain availability of pharmacological data for thousands of small molecules on 204 G protein-coupled receptors (GPCRs) provides a firm basis for an in-depth cross-pharmacology analysis of this superfamily. The number of protein targets included in the cross-pharmacology profile of the different GPCRs changes significantly upon varying the ligand similarity and binding affinity criteria. However, with the exception of muscarinic receptors, aminergic GPCRs distinguish themselves from the rest of the members in the family by their remarkably high levels of pharmacological similarity among them. Clusters of non-GPCR targets related by cross-pharmacology with particular GPCRs are identified and the implications for unwanted side-effects, as well as for repurposing opportunities, discussed. PMID:21851335

  2. CE-microreactor-CE-MS/MS for protein analysis

    PubMed Central

    Schoenherr, Regine M.; Ye, Mingliang; Vannatta, Michael

    2008-01-01

    We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome C and myoglobin generated Mascot MOWSE scores of 107 for cytochrome C and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively. PMID:17295444

  3. Human protein cluster analysis using amino acid frequencies.

    PubMed

    Vernone, Annamaria; Berchialla, Paola; Pescarmona, Gianpiero

    2013-01-01

    The paper focuses on the development of a software tool for protein clustering according to their amino acid content. All known human proteins were clustered according to the relative frequencies of their amino acids starting from the UniProtKB/Swiss-Prot reference database and making use of hierarchical cluster analysis. RESULTS were compared to those based on sequence similarities. Proteins display different clustering patterns according to type. Many extracellular proteins with highly specific and repetitive sequences (keratins, collagens etc.) cluster clearly confirming the accuracy of the clustering method. In our case clustering by sequence and amino acid content overlaps. Proteins with a more complex structure with multiple domains (catalytic, extracellular, transmembrane etc.), even if classified very similar according to sequence similarity and function (aquaporins, cadherins, steroid 5-alpha reductase etc.) showed different clustering according to amino acid content. Availability of essential amino acids according to local conditions (starvation, low or high oxygen, cell cycle phase etc.) may be a limiting factor in protein synthesis, whatever the mRNA level. This type of protein clustering may therefore prove a valuable tool in identifying so far unknown metabolic connections and constraints.

  4. Analysis of protein sequence/structure similarity relationships.

    PubMed Central

    Gan, Hin Hark; Perlow, Rebecca A; Roy, Sharmili; Ko, Joy; Wu, Min; Huang, Jing; Yan, Shixiang; Nicoletta, Angelo; Vafai, Jonathan; Sun, Ding; Wang, Lihua; Noah, Joyce E; Pasquali, Samuela; Schlick, Tamar

    2002-01-01

    Current analyses of protein sequence/structure relationships have focused on expected similarity relationships for structurally similar proteins. To survey and explore the basis of these relationships, we present a general sequence/structure map that covers all combinations of similarity/dissimilarity relationships and provide novel energetic analyses of these relationships. To aid our analysis, we divide protein relationships into four categories: expected/unexpected similarity (S and S(?)) and expected/unexpected dissimilarity (D and D(?)) relationships. In the expected similarity region S, we show that trends in the sequence/structure relation can be derived based on the requirement of protein stability and the energetics of sequence and structural changes. Specifically, we derive a formula relating sequence and structural deviations to a parameter characterizing protein stiffness; the formula fits the data reasonably well. We suggest that the absence of data in region S(?) (high structural but low sequence similarity) is due to unfavorable energetics. In contrast to region S, region D(?) (high sequence but low structural similarity) is well-represented by proteins that can accommodate large structural changes. Our analyses indicate that there are several categories of similarity relationships and that protein energetics provide a basis for understanding these relationships. PMID:12414710

  5. Global Analysis of Protein Expression of Inner Ear Hair Cells

    PubMed Central

    Wong, Ann C.Y.; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel

    2017-01-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes. SIGNIFICANCE STATEMENT Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that

  6. Comprehensive functional analysis of large lists of genes and proteins.

    PubMed

    Mlecnik, Bernhard; Galon, Jérôme; Bindea, Gabriela

    2017-03-22

    The interpretation of high dimensional datasets resulting from genomic and proteomic experiments in a timely and efficient manner is challenging. ClueGO software is a Cytoscape App that extracts representative functional biological information for large lists of genes or proteins. The functional enrichment analysis is based on the latest publicly available data from multiple annotation and ontology resources that can be automatically accessed through ClueGO. Predefined settings for the selection of the terms are provided to facilitate the analysis. Results are visualized as networks in which Gene Ontology (GO) terms and pathways are grouped based on their biological role. Many species are now supported by ClueGO and additional organisms are added on demand. ClueGO can be used together with the CluePedia App to enable the visualization of protein-protein interactions within or between pathways.

  7. Modern Analysis of Protein Folding by Differential Scanning Calorimetry.

    PubMed

    Ibarra-Molero, Beatriz; Naganathan, Athi N; Sanchez-Ruiz, Jose M; Muñoz, Victor

    2016-01-01

    Differential scanning calorimetry (DSC) is a very powerful tool for investigating protein folding and stability because its experimental output reflects the energetics of all conformations that become minimally populated during thermal unfolding. Accordingly, analysis of DSC experiments with simple thermodynamic models has been key for developing our understanding of protein stability during the past five decades. The discovery of ultrafast folding proteins, which have naturally broad conformational ensembles and minimally cooperative unfolding, opens the possibility of probing the complete folding free energy landscape, including those conformations at the top of the barrier to folding, via DSC. Exploiting this opportunity requires high-quality experiments and the implementation of novel analytical methods based on statistical mechanics. Here, we cover the recent exciting developments in this front, describing the new analytical procedures in detail as well as providing experimental guidelines for performing such analysis. © 2016 Elsevier Inc. All rights reserved.

  8. Protein complex analysis: From raw protein lists to protein interaction networks.

    PubMed

    Meysman, Pieter; Titeca, Kevin; Eyckerman, Sven; Tavernier, Jan; Goethals, Bart; Martens, Lennart; Valkenborg, Dirk; Laukens, Kris

    2017-09-01

    The elucidation of molecular interaction networks is one of the pivotal challenges in the study of biology. Affinity purification-mass spectrometry and other co-complex methods have become widely employed experimental techniques to identify protein complexes. These techniques typically suffer from a high number of false negatives and false positive contaminants due to technical shortcomings and purification biases. To support a diverse range of experimental designs and approaches, a large number of computational methods have been proposed to filter, infer and validate protein interaction networks from experimental pull-down MS data. Nevertheless, this expansion of available methods complicates the selection of the most optimal ones to support systems biology-driven knowledge extraction. In this review, we give an overview of the most commonly used computational methods to process and interpret co-complex results, and we discuss the issues and unsolved problems that still exist within the field. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:600-614, 2017. © 2015 Wiley Periodicals, Inc.

  9. THE APPLICATION OF MASS SPECTROMETRY TO PROTEIN ANALYSIS

    EPA Science Inventory

    The purpose of this presentation is to give our NHEERL collaborators a brief introduction to the use of mass spectrometric (MS) techniques in the analysis of proteins. The basic principles of electrospray ionization and matrix-assisted laser desorption ionization will be discuss...

  10. Educational Software for the Analysis of DNA and Protein Sequences.

    ERIC Educational Resources Information Center

    Maloy, Stanley; Olson, Sue

    1989-01-01

    Describes the development of the microcomputer-based educational software, DNAzoom, which was designed to introduce undergraduates in molecular biology to computer analysis of DNA protein sequences. Highlights include graphical presentation of data, the functional use of color, a menu-oriented interface, and students' evaluations of the software.…

  11. THE APPLICATION OF MASS SPECTROMETRY TO PROTEIN ANALYSIS

    EPA Science Inventory

    The purpose of this presentation is to give our NHEERL collaborators a brief introduction to the use of mass spectrometric (MS) techniques in the analysis of proteins. The basic principles of electrospray ionization and matrix-assisted laser desorption ionization will be discuss...

  12. Atom-by-atom analysis of global downhill protein folding

    NASA Astrophysics Data System (ADS)

    Sadqi, Mourad; Fushman, David; Muñoz, Victor

    2006-07-01

    Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.

  13. Bioinformatic analysis of the TonB protein family.

    PubMed

    Chu, Byron C H; Peacock, R Sean; Vogel, Hans J

    2007-06-01

    TonB is a protein prevalent in a large number of Gram-negative bacteria that is believed to be responsible for the energy transduction component in the import of ferric iron complexes and vitamin B(12) across the outer membrane. We have analyzed all the TonB proteins that are currently contained in the Entrez database and have identified nine different clusters based on its conserved 90-residue C-terminal domain amino acid sequence. The vast majority of the proteins contained a single predicted cytoplasmic transmembrane domain; however, nine of the TonB proteins encompass a approximately 290 amino acid N-terminal extension homologous to the MecR1 protein, which is composed of three additional predicted transmembrane helices. The periplasmic linker region, which is located between the N-terminal domain and the C-terminal domain, is extremely variable both in length (22-283 amino acids) and in proline content, indicating that a Pro-rich domain is not a required feature for all TonB proteins. The secondary structure of the C-terminal domain is found to be well preserved across all families, with the most variable region being between the second alpha-helix and the third beta-strand of the antiparallel beta-sheet. The fourth beta-strand found in the solution structure of the Escherichia coli TonB C-terminal domain is not a well conserved feature in TonB proteins in most of the clusters. Interestingly, several of the TonB proteins contained two C-terminal domains in series. This analysis provides a framework for future structure-function studies of TonB, and it draws attention to the unusual features of several TonB proteins.

  14. Proteomic Analysis of the Soybean Symbiosome Identifies New Symbiotic Proteins*

    PubMed Central

    Clarke, Victoria C.; Loughlin, Patrick C.; Gavrin, Aleksandr; Chen, Chi; Brear, Ella M.; Day, David A.; Smith, Penelope M.C.

    2015-01-01

    Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis. PMID

  15. Conformational diversity analysis reveals three functional mechanisms in proteins

    PubMed Central

    Fornasari, María Silvina

    2017-01-01

    Protein motions are a key feature to understand biological function. Recently, a large-scale analysis of protein conformational diversity showed a positively skewed distribution with a peak at 0.5 Å C-alpha root-mean-square-deviation (RMSD). To understand this distribution in terms of structure-function relationships, we studied a well curated and large dataset of ~5,000 proteins with experimentally determined conformational diversity. We searched for global behaviour patterns studying how structure-based features change among the available conformer population for each protein. This procedure allowed us to describe the RMSD distribution in terms of three main protein classes sharing given properties. The largest of these protein subsets (~60%), which we call “rigid” (average RMSD = 0.83 Å), has no disordered regions, shows low conformational diversity, the largest tunnels and smaller and buried cavities. The two additional subsets contain disordered regions, but with differential sequence composition and behaviour. Partially disordered proteins have on average 67% of their conformers with disordered regions, average RMSD = 1.1 Å, the highest number of hinges and the longest disordered regions. In contrast, malleable proteins have on average only 25% of disordered conformers and average RMSD = 1.3 Å, flexible cavities affected in size by the presence of disordered regions and show the highest diversity of cognate ligands. Proteins in each set are mostly non-homologous to each other, share no given fold class, nor functional similarity but do share features derived from their conformer population. These shared features could represent conformational mechanisms related with biological functions. PMID:28192432

  16. Cluster protein structures using recurrence quantification analysis on coordinates of alpha-carbon atoms of proteins

    NASA Astrophysics Data System (ADS)

    Zhou, Yu; Yu, Zu-Guo; Anh, Vo

    2007-08-01

    The 3-dimensional coordinates of alpha-carbon atoms of proteins are used to distinguish the protein structural classes based on recurrence quantification analysis (RQA). We consider two independent variables from RQA of coordinates of alpha-carbon atoms, %determ1 and %determ2, which were defined by Webber et al. [C.L. Webber Jr., A. Giuliani, J.P. Zbilut, A. Colosimo, Proteins Struct. Funct. Genet. 44 (2001) 292]. The variable %determ2 is used to define two new variables, %determ21 and %determ22. Then three variables %determ1, %determ21 and %determ22 are used to construct a 3-dimensional variable space. Each protein is represented by a point in this variable space. The points corresponding to proteins from the α, β, α+β and α/β structural classes position into different areas in this variable space. In order to give a quantitative assessment of our clustering on the selected proteins, Fisher's discriminant algorithm is used. Numerical results indicate that the discriminant accuracies are very high and satisfactory.

  17. Analysis of the fusion protein gene of the porcine rubulavirus LPMV: comparative analysis of paramyxovirus F proteins.

    PubMed

    Berg, M; Bergvall, A C; Svenda, M; Sundqvist, A; Moreno-López, J; Linné, T

    1997-01-01

    Complementary DNA clones representing the fusion (F) protein gene of the porcine rubulavirus LPMV were isolated and sequenced. The F gene was found to be 1,845 nucleotides long containing one long open reading frame capable of encoding a protein of 541 amino acids. The cleavage motif for F0 into F1 and F2 is His-Arg-Lys-Lys-Arg. A sequence comparison and a phylogenetic analysis was performed in order to identify possible functional domains of paramyxovirus fusion proteins and also to classify the porcine rubulavirus. The F gene of LPMV is most closely related to the human mumps virus and simian virus type 5 F genes, and is therefore classified into the rubulavirus genus. A coding region for a small hydrophobic protein was however not found between the F and hemagglutinin-neuraminidase (HN) genes as previously found in both SV5 and mumps.

  18. Chromatin immunoprecipitation and microarray-based analysis of protein location

    PubMed Central

    Lee, Tong Ihn; Johnstone, Sarah E; Young, Richard A

    2010-01-01

    Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types. PMID:17406303

  19. Functional and Structural Analysis of the Conserved EFhd2 Protein

    PubMed Central

    Acosta, Yancy Ferrer; Rodríguez Cruz, Eva N.; Vaquer, Ana del C.; Vega, Irving E.

    2013-01-01

    EFhd2 is a novel protein conserved from C. elegans to H. sapiens. This novel protein was originally identified in cells of the immune and central nervous systems. However, it is most abundant in the central nervous system, where it has been found associated with pathological forms of the microtubule-associated protein tau. The physiological or pathological roles of EFhd2 are poorly understood. In this study, a functional and structural analysis was carried to characterize the molecular requirements for EFhd2’s calcium binding activity. The results showed that mutations of a conserved aspartate on either EF-hand motif disrupted the calcium binding activity, indicating that these motifs work in pair as a functional calcium binding domain. Furthermore, characterization of an identified single-nucleotide polymorphisms (SNP) that introduced a missense mutation indicates the importance of a conserved phenylalanine on EFhd2 calcium binding activity. Structural analysis revealed that EFhd2 is predominantly composed of alpha helix and random coil structures and that this novel protein is thermostable. EFhd2’s thermo stability depends on its N-terminus. In the absence of the N-terminus, calcium binding restored EFhd2’s thermal stability. Overall, these studies contribute to our understanding on EFhd2 functional and structural properties, and introduce it into the family of canonical EF-hand domain containing proteins. PMID:22973849

  20. Proteomic analysis of protein methylation in the yeast Saccharomyces cerevisiae.

    PubMed

    Wang, Keyun; Zhou, Yongjin J; Liu, Hongwei; Cheng, Kai; Mao, Jiawei; Wang, Fangjun; Liu, Wujun; Ye, Mingliang; Zhao, Zongbao K; Zou, Hanfa

    2015-01-30

    Protein methylation catalyzed by SAM-dependent methyltransferase represents a major PTM involved in many important biological processes. Because methylation can occur on nitrogen, oxygen and sulfur centers and multiple methylation states exist on the nitrogen centers, methylproteome remains poorly documented. Here we present the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the yeast Saccharomyces cerevisiae based on the online multidimensional μHPLC/MS/MS technology. We identified 43 methylated proteins, containing 68 methylation events associated with 64 methylation sites. More than 90% of these methylation events were previously unannotated in Uniprot database. Our results indicated, 1) over 2.6% of identified S. cerevisiae proteins are methylated, 2) the amino acid residue preference of protein methylation follows the order Lys≫Arg>Asp>Asn≈Gln≈His>Glu>Cys, and 3) the methylation state on nitrogen center is largely exclusive. As our dataset covers various types of methylation centers, it provides rich information about yeast methylproteome and should significantly contribute to the field of protein methylation. In this paper, we presented the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the yeast S. cerevisiae and collected a comprehensive list of proteins methylated on a set of distinct residues (K, R, N, E, D, Q, H, C). Our study provided useful information about the amino acid residue preference and methylation state distributions on nitrogen centers of protein methylation in S. cerevisiae. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  2. Prediction of Hydrophobic Cores of Proteins Using Wavelet Analysis.

    PubMed

    Hirakawa; Kuhara

    1997-01-01

    Information concerning the secondary structures, flexibility, epitope and hydrophobic regions of amino acid sequences can be extracted by assigning physicochemical indices to each amino acid residue, and information on structure can be derived using the sliding window averaging technique, which is in wide use for smoothing out raw functions. Wavelet analysis has shown great potential and applicability in many fields, such as astronomy, radar, earthquake prediction, and signal or image processing. This approach is efficient for removing noise from various functions. Here we employed wavelet analysis to smooth out a plot assigned to a hydrophobicity index for amino acid sequences. We then used the resulting function to predict hydrophobic cores in globular proteins. We calculated the prediction accuracy for the hydrophobic cores of 88 representative set of proteins. Use of wavelet analysis made feasible the prediction of hydrophobic cores at 6.13% greater accuracy than the sliding window averaging technique.

  3. Objective Diagnosis of Cervical Cancer by Tissue Protein Profile Analysis

    NASA Astrophysics Data System (ADS)

    Patil, Ajeetkumar; Bhat, Sujatha; Rai, Lavanya; Kartha, V. B.; Chidangil, Santhosh

    2011-07-01

    Protein profiles of homogenized normal cervical tissue samples from hysterectomy subjects and cancerous cervical tissues from biopsy samples collected from patients with different stages of cervical cancer were recorded using High Performance Liquid Chromatography coupled with Laser Induced Fluorescence (HPLC-LIF). The Protein profiles were subjected to Principle Component Analysis to derive statistically significant parameters. Diagnosis of sample types were carried out by matching three parameters—scores of factors, squared residuals, and Mahalanobis Distance. ROC and Youden's Index curves for calibration standards were used for objective estimation of the optimum threshold for decision making and performance.

  4. Circular Dichroism for the Analysis of Protein-DNA Interactions.

    PubMed

    Scarlett, Garry; Siligardi, Giuliano; Kneale, Geoffrey G

    2015-01-01

    The aim of this chapter is to provide information on the practical aspects of circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD) in protein-nucleic acids interaction solution studies. The chapter will describe the guidelines appropriate to designing experiments and conducting correct data interpretation, the use of both benchtop and synchrotron CD approaches is discussed and the advantages of SRCD outlined. Further information and a good general review of the field a can be found in Gray (Circular Dichroism of protein-nucleic acid interactions. In: Fasman GD (ed) Circular dichroism and the conformational analysis of biomolecules. Plenum Press, New York. pp 469-500, 1996).

  5. Patterns in protein primary sequences: classification, display and analysis.

    PubMed Central

    Saurugger, P. N.; Metfessel, B. A.

    1991-01-01

    The protein folding code, which is contained in the amino acid chain of a protein, has so far eluded elucidation. However, patterns of hydrophobic residues have previously been identified which show a specificity towards certain secondary structural elements. We are developing an analysis toolkit to find, visualize, and analyze patterns in primary sequences. Preliminary results show that there exist patterns in primary sequences which are useful for predicting the structural class of amino acid chains, performing especially well for the all-alpha helix and all-beta sheet classes. PMID:1807631

  6. Quantitative analysis of protein-ligand interactions by NMR.

    PubMed

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  7. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    PubMed

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

  8. A protein extraction method for low protein concentration solutions compatible with the proteomic analysis of rubber particles.

    PubMed

    Wang, Dan; Sun, Yong; Tong, Zheng; Yang, Qian; Chang, Lili; Meng, Xueru; Wang, Limin; Tian, Weimin; Wang, Xuchu

    2016-11-01

    The extraction of high-purity proteins from the washing solution (WS) of rubber particles (also termed latex-producing organelles) from laticifer cells in rubber tree for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high-purity WS proteins. We improved our current borax and phenol-based method by adding reextraction steps with phenol (REP) to improve the yield from low protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original borax and phenol-based method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high-quality from starting samples containing less than 0.02 mg of proteins per milliliter. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP-extracted WS proteins were resolved by 2DE, and 28 proteins were positively identified by MS. This method has the potential to become widely used for the extraction of proteins from low protein concentration solutions for proteomic analysis.

  9. N-terminal sequence analysis of proteins and peptides.

    PubMed

    Reim, D F; Speicher, D W

    2001-05-01

    Amino-terminal (N-terminal) sequence analysis is used to identify the order of amino acids of proteins or peptides, starting at their N-terminal end. This unit describes the sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Perkin-Elmer Procise Sequencer. Sequence analysis of protein or peptide samples in solution or bound to PVDF membranes using a Hewlett-Packard Model G1005A sequencer is also described. Methods are provided for optimizing separation of PTH amino acid derivatives on Perkin-Elmer instruments and for increasing the proportion of sample injected onto the PTH analyzer on older Perkin-Elmer instruments by installing a modified sample loop. The amount of data obtained from a single sequencer run is substantial, and careful interpretation of this data by an experienced scientist familiar with the current operation performance of the instrument used for this analysis is critically important. A discussion of data interpretation is therefore provided. Finally, discussion of optimization of sequencer performance as well as possible solutions to frequently encountered problems is included.

  10. Spectroscopic analysis of protein Fe-NO complexes.

    PubMed

    Bellota-Antón, César; Munnoch, John; Robb, Kirsty; Adamczyk, Katrin; Candelaresi, Marco; Parker, Anthony W; Dixon, Ray; Hutchings, Matthew I; Hunt, Neil T; Tucker, Nicholas P

    2011-10-01

    The toxic free radical NO (nitric oxide) has diverse biological roles in eukaryotes and bacteria, being involved in signalling, vasodilation, blood clotting and immunity, and as an intermediate in microbial denitrification. The predominant biological mechanism of detecting NO is through the formation of iron nitrosyl complexes, although this is a deleterious process for other iron-containing enzymes. We have previously applied techniques such as UV-visible and EPR spectroscopy to the analysis of protein Fe-NO complex formation in order to study how NO controls the activity of the bacterial transcriptional regulators NorR and NsrR. These studies have analysed NO-dependent biological activity both in vitro and in vivo using diverse biochemical, molecular and spectroscopic methods. Recently, we have applied ultrafast 2D-IR (two-dimensional IR) spectroscopy to the analysis of NO-protein interactions using Mb (myoglobin) and Cc (cytochrome c) as model haem proteins. The ultrafast fluctuations of Cc and Mb show marked differences, indicating altered flexibility of the haem pockets. We have extended this analysis to bacterial catalase enzymes that are known to play a role in the nitrosative stress response by detoxifying peroxynitrite. The first 2D-IR analysis of haem nitrosylation and perspectives for the future are discussed.

  11. Network analysis reveals common host protein/s modulating pathogenesis of neurotropic viruses

    PubMed Central

    Ghosh, Sourish; Mukherjee, Sriparna; Sengupta, Nabonita; Roy, Arunava; Dey, Dhritiman; Chakraborty, Surajit; Chattopadhyay, Dhrubajyoti; Banerjee, Arpan; Basu, Anirban

    2016-01-01

    Network analysis through graph theory provides a quantitative approach to characterize specific proteins and their constituent assemblies that underlie host-pathogen interactions. In the present study, graph theory was used to analyze the interactome designed out of 50 differentially expressing proteins from proteomic analysis of Chandipura Virus (CHPV, Family: Rhabdoviridae) infected mouse brain tissue to identify the primary candidates for intervention. Using the measure of degree centrality, that quantifies the connectedness of a single protein within a milieu of several other interacting proteins, DJ-1 was selected for further molecular validation. To elucidate the generality of DJ-1’s role in propagating infection its role was also monitored in another RNA virus, Japanese Encephalitis Virus (JEV, Family: Flaviviridae) infection. Concurrently, DJ-1 got over-expressed in response to reactive oxygen species (ROS) generation following viral infection which in the early phase of infection migrated to mitochondria to remove dysfunctional mitochondria through the process of mitophagy. DJ-1 was also observed to modulate the viral replication and interferon responses along with low-density lipoprotein (LDL) receptor expression in neurons. Collectively these evidences reveal a comprehensive role for DJ-1 in neurotropic virus infection in the brain. PMID:27581498

  12. Analysis of the interface variability in NMR structure ensembles of protein-protein complexes.

    PubMed

    Calvanese, Luisa; D'Auria, Gabriella; Vangone, Anna; Falcigno, Lucia; Oliva, Romina

    2016-06-01

    NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html.

  13. Calculations of electrostatic properties in proteins. Analysis of contributions from induced protein dipoles.

    PubMed

    Van Belle, D; Couplet, I; Prevost, M; Wodak, S J

    1987-12-20

    The calculation of induced dipole moments and of their contribution to electrostatic effects in proteins is implemented following the approach of Warshel. Isotropic polarizabilities are assigned to individual atoms, and the resulting deviation from pairwise interactions is treated by a self-consistent iterative procedure. We give a detailed description of how the formalism is implemented in molecular mechanics and molecular dynamics simulation procedures, and report results based on calculations performed on crystal structures of crambin, liver alcohol dehydrogenase and ribonuclease T1. We focus our analysis on evaluating the contribution of polarizability of the protein matrix to electrostatic energies, local fields, to dipole moments of peptide groups and of secondary structure elements in the polypeptide chain. Our calculations confirm that induced dipole moments in proteins provide important stabilizing contributions to electrostatic energies, and that these contributions cannot be mimicked by the usual approximations where either a continuum dielectric constant, or a distance-dependent dielectric function is used. We find that induced protein dipoles appreciably affect the magnitude and direction of local electrostatic fields in a manner that is strongly influenced by the microscopic environment in the protein. Most strongly affected are fields in charged groups that are involved in close interactions with other charged groups, while the influence on local fields of aliphatic groups is marginal. We find, moreover, that induction effects from surrounding protein atoms tend on average to increase peptide dipoles and helix macro-dipoles by about 16%, again reflecting electrostatic stabilization by the protein matrix, and show that (at least in the alpha/beta domain of alcohol dehydrogenase) the contribution of side-chains to this stabilization is significant.

  14. Protein-protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection.

    PubMed

    Benz, Christian; Retzbach, Heiko; Nagl, Stefan; Belder, Detlev

    2013-07-21

    Herein, we demonstrate the feasibility of a protein-protein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft- and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of 7.9 and 0.75 Hz, respectively. They were used for detection of protein-protein interactions in microdroplets using a model system of Alexa Fluor 488 labelled biotinylated BSA, Alexa Fluor 594 labelled streptavidin and unlabelled chicken egg white avidin. These microchips could be used for quantitative detection of avidin and streptavidin in microdroplets in direct and competitive assay formats with nanomolar detection limits, corresponding to attomole protein amounts. Four droplets were found to be sufficient for analytical determination. Fluorescence intensity ratio and fluorescence lifetime measurements were performed and compared for microdroplet FRET determination. A competitive on-chip binding assay for determination of unlabelled avidin using fluorescence lifetime detection could be performed within 135 s only.

  15. A comprehensive analysis of the La-motif protein superfamily

    PubMed Central

    Bousquet-Antonelli, Cécile; Deragon, Jean-Marc

    2009-01-01

    The extremely well-conserved La motif (LAM), in synergy with the immediately following RNA recognition motif (RRM), allows direct binding of the (genuine) La autoantigen to RNA polymerase III primary transcripts. This motif is not only found on La homologs, but also on La-related proteins (LARPs) of unrelated function. LARPs are widely found amongst eukaryotes and, although poorly characterized, appear to be RNA-binding proteins fulfilling crucial cellular functions. We searched the fully sequenced genomes of 83 eukaryotic species scattered along the tree of life for the presence of LAM-containing proteins. We observed that these proteins are absent from archaea and present in all eukaryotes (except protists from the Plasmodium genus), strongly suggesting that the LAM is an ancestral motif that emerged early after the archaea-eukarya radiation. A complete evolutionary and structural analysis of these proteins resulted in their classification into five families: the genuine La homologs and four LARP families. Unexpectedly, in each family a conserved domain representing either a classical RRM or an RRM-like motif immediately follows the LAM of most proteins. An evolutionary analysis of the LAM-RRM/RRM-L regions shows that these motifs co-evolved and should be used as a single entity to define the functional region of interaction of LARPs with their substrates. We also found two extremely well conserved motifs, named LSA and DM15, shared by LARP6 and LARP1 family members, respectively. We suggest that members of the same family are functional homologs and/or share a common molecular mode of action on different RNA baits. PMID:19299548

  16. A comprehensive analysis of the La-motif protein superfamily.

    PubMed

    Bousquet-Antonelli, Cécile; Deragon, Jean-Marc

    2009-05-01

    The extremely well-conserved La motif (LAM), in synergy with the immediately following RNA recognition motif (RRM), allows direct binding of the (genuine) La autoantigen to RNA polymerase III primary transcripts. This motif is not only found on La homologs, but also on La-related proteins (LARPs) of unrelated function. LARPs are widely found amongst eukaryotes and, although poorly characterized, appear to be RNA-binding proteins fulfilling crucial cellular functions. We searched the fully sequenced genomes of 83 eukaryotic species scattered along the tree of life for the presence of LAM-containing proteins. We observed that these proteins are absent from archaea and present in all eukaryotes (except protists from the Plasmodium genus), strongly suggesting that the LAM is an ancestral motif that emerged early after the archaea-eukarya radiation. A complete evolutionary and structural analysis of these proteins resulted in their classification into five families: the genuine La homologs and four LARP families. Unexpectedly, in each family a conserved domain representing either a classical RRM or an RRM-like motif immediately follows the LAM of most proteins. An evolutionary analysis of the LAM-RRM/RRM-L regions shows that these motifs co-evolved and should be used as a single entity to define the functional region of interaction of LARPs with their substrates. We also found two extremely well conserved motifs, named LSA and DM15, shared by LARP6 and LARP1 family members, respectively. We suggest that members of the same family are functional homologs and/or share a common molecular mode of action on different RNA baits.

  17. Preliminary crystallographic analysis of the bacteriophage P22 portal protein.

    PubMed

    Cingolani, Gino; Moore, Sean D; Prevelige, Peter E; Johnson, John E

    2002-07-01

    Portal proteins are components of large oligomeric dsDNA pumps connecting the icosahedral capsid of tailed bacteriophages to the tail. Prior to the tail attachment, dsDNA is actively pumped through a central cavity formed by the subunits. We have studied the portal protein of bacteriophage P22, which is the largest connector characterized among the tailed bacteriophages. The molecular weight of the monomer is 82.7 kDa, and it spontaneously assembles into an oligomeric structure of approximately 1.0 MDa. Here we present a preliminary biochemical and crystallographic characterization of this large macromolecular complex. The main difficulties related to the crystallization of P22 portal protein lay in the intrinsic dynamic nature of the portal oligomer. Recombinant connectors assembled from portal monomers expressed in Escherichia coli form rings of different stoichiometry in solution, which cannot be separated on the basis of their size. To overcome this intrinsic heterogeneity we devised a biochemical purification that separates different ring populations on the basis of their charge. Small ordered crystals were grown from drops containing a high concentration of the kosmotropic agent tert-butanol and used for data collection. A preliminary crystallographic analysis to 7.0-A resolution revealed that the P22 portal protein crystallized in space group I4 with unit cell dimensions a=b=409.4A, c=260.4A. This unit cell contains a total of eight connectors. Analysis of the noncrystallographic symmetry by the self-rotation function unambiguously confirmed that bacteriophage P22 portal protein is a dodecamer with a periodicity of 30 degrees. The cryo-EM reconstruction of the dodecahedral bacteriophage T3 portal protein will be used as a model to initiate phase extension and structure determination.

  18. Analysis of proteins by particle induced X-ray emission

    NASA Astrophysics Data System (ADS)

    Natal da Luz, H.; Spemann, D.; Meyer-Klaucke, W.; Tröger, W.

    2005-04-01

    The development of a standard PIXE procedure dedicated to the metal analysis in proteins is of great interest for biochemists. In order to achieve this aim, the LIPSION microprobe facility at the Leipzig University was used with a 2.25 MeV scanning proton micro-beam for the investigation of several test systems with known metal concentrations and stoichiometries. STIM and RBS were performed in these systems to select the best technique for areal density determination. With this set-up STIM proved to be less sensitive to sample thickness inhomogeneities. Radiation damage was evaluated in real proteins, such as glyoxalase II and no effects were noticed for currents up to 500 pA. Glyoxalase II served also to demonstrate the ability of PIXE to determine the metal stoichiometry even for protein concentrations of ∼60 μM. The zinc phosphodiesterase and the flavorubredoxin lactamase domain were also analyzed. The use of PIXE, combined with STIM for areal density determination, turned out to be a very powerful technique in protein analysis, allowing for stoichiometry determinations of very small sample quantities without any further chemical preparation.

  19. Capillary isoelectric focusing-electrospray mass spectrometry for protein analysis

    SciTech Connect

    Tang, Q.; Harrata, A.K.; Lee, C.S.

    1995-10-01

    On-line combination of capillary isoelectric focusing (CIEF) with electrospray mass spectrometry (ESMS) as a two-dimensional separation system is demonstrated. Mixtures of model proteins including cytochrome c (horse heart), myoglobin (horse heart), and carbonic anhydrase II (bovine erythrocyte) are focused and cathodically mobilized in a polyacrylamide-coated capillary. At the end of CIEF capillary, the mobilized protein zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow configuration. The effects of carrier ampholyte concentration on the CIEF separation and the protein electrospray ionization mass spectra are presented and discussed. In this study, the focusing effect of CIEF permits analysis of very dilute protein samples. A typical concentration factor of 50-100 times is observed. The concentration detection limit of myoglobin for a full-scan CIEF-ESMS analysis is in the range of 10{sup -7} M, 2 orders of magnitude over that possible with normal capillary zone electrophoresis ESMS. 35 refs., 5 figs., 2 tabs.

  20. A Fourier analysis of symmetry in protein structure.

    PubMed

    Taylor, William R; Heringa, Jaap; Baud, Franck; Flores, Tomas P

    2002-02-01

    The score matrix from a structure comparison program (SAP) was used to search for repeated structures using a Fourier analysis. When tested with artificial data, a simple Fourier transform of the smoothed matrix provided a clear signal of the repeat periodicity that could be used to extract the repeating units with the SAP program. The strength of the Fourier signal was calibrated against the signal from model proteins. The most useful of these was the novel random-walk approach employed to generate realistic 'fake' structures. On the basis of these it was possible to conclude that only a small proportion of protein structures have an unexpected degree of symmetry. Artificially generated 'ideal' folds provided an upper limit on the strength of signal that could be expected from a 'perfectly' repeating compact structure. Unexpectedly, some of the very regular beta-propellor folds attained the same strength but the majority of symmetric structures lay below this region. When native proteins were ranked by the power of their spectrum a wide variety of fold types were seen to score highly. In the betaalpha class, these included the globular betaalpha proteins and the more repetitive leucine-rich betaalpha folds. In the all-beta class; beta-propellors, beta-prisms and beta-helices were found as well as the more globular gamma-crystalin domains. When this ranked list was filtered to remove proteins that contained detectable internal sequence similarity (using the program REPRO), the list became exclusively composed of just globular betaalpha class proteins and in the top 50 re-ranked proteins, only a single 4-fold propellor structure remained.

  1. Protein pharmacophore selection using hydration-site analysis

    PubMed Central

    Hu, Bingjie; Lill, Markus A.

    2012-01-01

    Virtual screening using pharmacophore models is an efficient method to identify potential lead compounds for target proteins. Pharmacophore models based on protein structures are advantageous because a priori knowledge of active ligands is not required and the models are not biased by the chemical space of previously identified actives. However, in order to capture most potential interactions between all potentially binding ligands and the protein, the size of the pharmacophore model, i.e. number of pharmacophore elements, is typically quite large and therefore reduces the efficiency of pharmacophore based screening. We have developed a new method to select important pharmacophore elements using hydration-site information. The basic premise is that ligand functional groups that replace water molecules in the apo protein contribute strongly to the overall binding affinity of the ligand, due to the additional free energy gained from releasing the water molecule into the bulk solvent. We computed the free energy of water released from the binding site for each hydration site using thermodynamic analysis of molecular dynamics (MD) simulations. Pharmacophores which are co-localized with hydration sites with estimated favorable contributions to the free energy of binding are selected to generate a reduced pharmacophore model. We constructed reduced pharmacophore models for three protein systems and demonstrated good enrichment quality combined with high efficiency. The reduction in pharmacophore model size reduces the required screening time by a factor of 200–500 compared to using all protein pharmacophore elements. We also describe a training process using a small set of known actives to reliably select the optimal set of criteria for pharmacophore selection for each protein system. PMID:22397751

  2. Computer-based analysis of Haemophilus parasuis protein fingerprints

    PubMed Central

    2004-01-01

    Abstract The present study aimed to compare the whole-cell protein profiles of Haemophilus parasuis field isolates by using a computer-based analysis, and evaluate the relationship between polyacrylamide gel electrophoresis (PAGE) type and virulence potential based on isolation site. A dendrogram clustering isolates with similar protein profiles was generated. Haemophilus parasuis isolates were grouped into 2 major PAGE type groups. The PAGE type II isolates were characterized by the presence of major proteins with molecular weights varying from between 36 and 38 kDa and included 90.7% of the isolates recovered from systemic sites, such as pleura, pericardium, peritoneum, lymph nodes, joints, and brain. Isolates classified as PAGE type I were characterized by the absence of this group of proteins and included 83.4% of the isolates recovered from the upper respiratory tract of healthy animals. The present study further corroborates the existence of a unique group of major proteins in potentially virulent H. parasuis isolates. PMID:14979439

  3. N-terminal protein processing: A comparative proteogenomic analysis

    SciTech Connect

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  4. Analysis of protein dynamics in the pericellular matrix

    NASA Astrophysics Data System (ADS)

    Scrimgeour, Jan; Young, Dylan

    2015-03-01

    The pericellular matrix (PCM) is a low density, hydrated polymer coating that extends into the extracellular space from the surface of many living cells. The PCM controls access to cell and tissue surfaces, regulating a diverse set of processes from cell adhesion to protein transport and storage. The cell coat consists of a malleable backbone - the large polysaccharide hyaluronan (HA) - with its structure, its material properties, and its bio-functionality tuned by a diverse set of HA binding proteins. These proteins add charge, cross-links and growth factor-like ligands into the brush. Dynamic interactions between the HA and its binding proteins can be observed using single particle tracking in a fluorescence microscope. The resulting single molecule trajectories can contain evidence of site hoping, with the proteins dynamically moving between different states of motion as they bind and unbind from the HA. Here, we present an evaluation of hidden Markov models for the analysis of such multi-mobility trajectories. Simulated trajectories are used to probe the limits of this approach for molecular trajectories of limited length and the results are used to inform the design of particle tracking experiments.

  5. Analysis of informational redundancy in the protein-assembling machinery

    NASA Astrophysics Data System (ADS)

    Berkovich, Simon

    2004-03-01

    Entropy analysis of the DNA structure does not reveal a significant departure from randomness indicating lack of informational redundancy. This signifies the absence of a hidden meaning in the genome text and supports the 'barcode' interpretation of DNA given in [1]. Lack of informational redundancy is a characteristic property of an identification label rather than of a message of instructions. Yet randomness of DNA has to induce non-random structures of the proteins. Protein synthesis is a two-step process: transcription into RNA with gene splicing and formation a structure of amino acids. Entropy estimations, performed by A. Djebbari, show typical values of redundancy of the biomolecules along these pathways: DNA gene 4proteins 15-40in gene expression, the RNA copy carries the same information as the original DNA template. Randomness is essentially eliminated only at the step of the protein creation by a degenerate code. According to [1], the significance of the substitution of U for T with a subsequent gene splicing is that these transformations result in a different pattern of RNA oscillations, so the vital DNA communications are protected against extraneous noise coming from the protein making activities. 1. S. Berkovich, "On the 'barcode' functionality of DNA, or the Phenomenon of Life in the Physical Universe", Dorrance Publishing Co., Pittsburgh, 2003

  6. Analysis of proteins involved in biodegradation of crop biomass

    NASA Technical Reports Server (NTRS)

    Crawford, Kamau; Trotman, Audrey

    1998-01-01

    The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

  7. Analysis of proteins involved in biodegradation of crop biomass

    NASA Technical Reports Server (NTRS)

    Crawford, Kamau; Trotman, Audrey

    1998-01-01

    The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

  8. Kinetic Analysis of Protein Crystal Nucleation in Gel Matrix

    PubMed Central

    Wang, Lei; Liu, Xiang-Yang

    2008-01-01

    The effect of agarose on nucleation of hen egg white lysozyme crystal was examined quantitatively using a temperature-jumping technique. For the first time, to our knowledge, the inhibition of agarose during the nucleation of lysozyme was quantified in two respects: a), the effect of increasing interfacial nucleation barrier, described by the so-called interfacial correlation parameter f(m); and b), the ratio of diffusion to interfacial kinetics obtained from dynamic surface tension measurements. It follows from a dynamic surface tension analysis that the agarose network inhibits the nucleation of lysozyme by means of an enhancement of the repulsion and interfacial structure mismatch between foreign bodies and lysozyme crystals, slowing down the diffusion process of the protein molecules and clusters toward the crystal-fluid interface and inhibiting the rearrangement of protein molecules at the interface. Our results, based on ultraviolet-visible spectroscopy, also show no evidence of the supersaturation enhancement effect in protein agarose gels. The effects of nucleation suppression and transport limitation in gels result in bigger, fewer, and perhaps better quality protein crystals. The understandings obtained in this study will improve our knowledge in controlling the crystallization of proteins and other biomolecules. PMID:18835910

  9. Fluorescent protein recruitment assay for demonstration and analysis of in vivo protein interactions in plant cells and its application to Tobacco mosaic virus movement protein.

    PubMed

    Boutant, Emmanuel; Didier, Pascal; Niehl, Annette; Mély, Yves; Ritzenthaler, Christophe; Heinlein, Manfred

    2010-04-01

    We describe a simple fluorescent protein-based method to investigate interactions with a viral movement protein in living cells that relies on the in vivo re-localization of proteins in the presence of their interaction partners. We apply this method in combination with fluorescence lifetime imaging microscopy (FLIM) to demonstrate that a domain of the Tobacco mosaic virus (TMV) movement protein (MP) previously predicted to mediate protein:protein interactions is dispensable for these contacts. We suggest that this method can be generalized for analysis of other protein interactions in planta.

  10. Removal of detergents from protein digests for mass spectrometry analysis

    PubMed Central

    Yeung, Yee-Guide; Nieves, Edward; Angeletti, Ruth; Stanley, E. Richard

    2008-01-01

    Detergents are commonly used for the extraction of hydrophobic proteins and must be removed for sensitive detection of peptides by mass spectrometry (MS). We demonstrate that ethyl acetate (EA) is able to extract octylglycoside (OG) from a protease digest without loss of peptides or interference with the MS peptide spectral profile. EA extraction was also found to reduce interference of SDS, NP-40 or Triton X-100 in the MS analysis. PMID:18713617

  11. Analysis and Ranking of Protein-Protein Docking Models Using Inter-Residue Contacts and Inter-Molecular Contact Maps.

    PubMed

    Oliva, Romina; Chermak, Edrisse; Cavallo, Luigi

    2015-07-01

    In view of the increasing interest both in inhibitors of protein-protein interactions and in protein drugs themselves, analysis of the three-dimensional structure of protein-protein complexes is assuming greater relevance in drug design. In the many cases where an experimental structure is not available, protein-protein docking becomes the method of choice for predicting the arrangement of the complex. However, reliably scoring protein-protein docking poses is still an unsolved problem. As a consequence, the screening of many docking models is usually required in the analysis step, to possibly single out the correct ones. Here, making use of exemplary cases, we review our recently introduced methods for the analysis of protein complex structures and for the scoring of protein docking poses, based on the use of inter-residue contacts and their visualization in inter-molecular contact maps. We also show that the ensemble of tools we developed can be used in the context of rational drug design targeting protein-protein interactions.

  12. Towards accurate modeling of noncovalent interactions for protein rigidity analysis.

    PubMed

    Fox, Naomi; Streinu, Ileana

    2013-01-01

    Protein rigidity analysis is an efficient computational method for extracting flexibility information from static, X-ray crystallography protein data. Atoms and bonds are modeled as a mechanical structure and analyzed with a fast graph-based algorithm, producing a decomposition of the flexible molecule into interconnected rigid clusters. The result depends critically on noncovalent atomic interactions, primarily on how hydrogen bonds and hydrophobic interactions are computed and modeled. Ongoing research points to the stringent need for benchmarking rigidity analysis software systems, towards the goal of increasing their accuracy and validating their results, either against each other and against biologically relevant (functional) parameters. We propose two new methods for modeling hydrogen bonds and hydrophobic interactions that more accurately reflect a mechanical model, without being computationally more intensive. We evaluate them using a novel scoring method, based on the B-cubed score from the information retrieval literature, which measures how well two cluster decompositions match. To evaluate the modeling accuracy of KINARI, our pebble-game rigidity analysis system, we use a benchmark data set of 20 proteins, each with multiple distinct conformations deposited in the Protein Data Bank. Cluster decompositions for them were previously determined with the RigidFinder method from Gerstein's lab and validated against experimental data. When KINARI's default tuning parameters are used, an improvement of the B-cubed score over a crude baseline is observed in 30% of this data. With our new modeling options, improvements were observed in over 70% of the proteins in this data set. We investigate the sensitivity of the cluster decomposition score with case studies on pyruvate phosphate dikinase and calmodulin. To substantially improve the accuracy of protein rigidity analysis systems, thorough benchmarking must be performed on all current systems and future

  13. Towards accurate modeling of noncovalent interactions for protein rigidity analysis

    PubMed Central

    2013-01-01

    Background Protein rigidity analysis is an efficient computational method for extracting flexibility information from static, X-ray crystallography protein data. Atoms and bonds are modeled as a mechanical structure and analyzed with a fast graph-based algorithm, producing a decomposition of the flexible molecule into interconnected rigid clusters. The result depends critically on noncovalent atomic interactions, primarily on how hydrogen bonds and hydrophobic interactions are computed and modeled. Ongoing research points to the stringent need for benchmarking rigidity analysis software systems, towards the goal of increasing their accuracy and validating their results, either against each other and against biologically relevant (functional) parameters. We propose two new methods for modeling hydrogen bonds and hydrophobic interactions that more accurately reflect a mechanical model, without being computationally more intensive. We evaluate them using a novel scoring method, based on the B-cubed score from the information retrieval literature, which measures how well two cluster decompositions match. Results To evaluate the modeling accuracy of KINARI, our pebble-game rigidity analysis system, we use a benchmark data set of 20 proteins, each with multiple distinct conformations deposited in the Protein Data Bank. Cluster decompositions for them were previously determined with the RigidFinder method from Gerstein's lab and validated against experimental data. When KINARI's default tuning parameters are used, an improvement of the B-cubed score over a crude baseline is observed in 30% of this data. With our new modeling options, improvements were observed in over 70% of the proteins in this data set. We investigate the sensitivity of the cluster decomposition score with case studies on pyruvate phosphate dikinase and calmodulin. Conclusion To substantially improve the accuracy of protein rigidity analysis systems, thorough benchmarking must be performed on all

  14. [Structure analysis of disease-related proteins using vibrational spectroscopy].

    PubMed

    Hiramatsu, Hirotsugu

    2014-01-01

    Analyses of the structure and properties of identified pathogenic proteins are important for elucidating the molecular basis of diseases and in drug discovery research. Vibrational spectroscopy has advantages over other techniques in terms of sensitivity of detection of structural changes. Spectral analysis, however, is complicated because the spectrum involves a substantial amount of information. This article includes examples of structural analysis of disease-related proteins using vibrational spectroscopy in combination with additional techniques that facilitate data acquisition and analysis. Residue-specific conformation analysis of an amyloid fibril was conducted using IR absorption spectroscopy in combination with (13)C-isotope labeling, linear dichroism measurement, and analysis of amide I band features. We reveal a pH-dependent property of the interacting segment of an amyloidogenic protein, β2-microglobulin, which causes dialysis-related amyloidosis. We also reveal the molecular mechanisms underlying pH-dependent sugar-binding activity of human galectin-1, which is involved in cell adhesion, using spectroscopic techniques including UV resonance Raman spectroscopy. The decreased activity at acidic pH was attributed to a conformational change in the sugar-binding pocket caused by protonation of His52 (pKa 6.3) and the cation-π interaction between Trp68 and the protonated His44 (pKa 5.7). In addition, we show that the peak positions of the Raman bands of the C4=C5 stretching mode at approximately 1600 cm(-1) and the Nπ-C2-Nτ bending mode at approximately 1405 cm(-1) serve as markers of the His side-chain structure. The Raman signal was enhanced 12 fold using a vertical flow apparatus.

  15. Quantitative Analysis of Single-Molecule RNA-Protein Interaction

    PubMed Central

    Fuhrmann, Alexander; Schoening, Jan C.; Anselmetti, Dario; Staiger, Dorothee; Ros, Robert

    2009-01-01

    Abstract RNA-binding proteins impact gene expression at the posttranscriptional level by interacting with cognate cis elements within the transcripts. Here, we apply dynamic single-molecule force spectroscopy to study the interaction of the Arabidopsis glycine-rich RNA-binding protein AtGRP8 with its RNA target. A dwell-time-dependent analysis of the single-molecule data in combination with competition assays and site-directed mutagenesis of both the RNA target and the RNA-binding domain of the protein allowed us to distinguish and quantify two different binding modes. For dwell times <0.21 s an unspecific complex with a lifetime of 0.56 s is observed, whereas dwell times >0.33 s result in a specific interaction with a lifetime of 208 s. The corresponding reaction lengths are 0.28 nm for the unspecific and 0.55 nm for the specific AtGRP8-RNA interactions, indicating formation of a tighter complex with increasing dwell time. These two binding modes cannot be dissected in ensemble experiments. Quantitative titration in RNA bandshift experiments yields an ensemble-averaged equilibrium constant of dissociation of KD = 2 × 10−7 M. Assuming comparable on-rates for the specific and nonspecific binding modes allows us to estimate their free energies as ΔG0 = −42 kJ/mol and ΔG0 = −28 kJ/mol for the specific and nonspecific binding modes, respectively. Thus, we show that single-molecule force spectroscopy with a refined statistical analysis is a potent tool for the analysis of protein-RNA interactions without the drawback of ensemble averaging. This makes it possible to discriminate between different binding modes or sites and to analyze them quantitatively. We propose that this method could be applied to complex interactions of biomolecules in general, and be of particular interest for the investigation of multivalent binding reactions. PMID:19527663

  16. Predicting human plasma protein binding of drugs using plasma protein interaction QSAR analysis (PPI-QSAR).

    PubMed

    Li, Haiyan; Chen, Zhuxi; Xu, Xuejun; Sui, Xiaofan; Guo, Tao; Liu, Wei; Zhang, Jiwen

    2011-09-01

    A novel method, named as the plasma protein-interaction QSAR analysis (PPI-QSAR) was used to construct the QSAR models for human plasma protein binding. The intra-molecular descriptors of drugs and inter-molecular interaction descriptors resulted from the docking simulation between drug molecules and human serum albumin were included as independent variables in this method. A structure-based in silico model for a data set of 65 antibiotic drugs was constructed by the multiple linear regression method and validated by the residual analysis, the normal Probability-Probability plot and Williams plot. The R(2) and Q(2) values of the entire data set were 0.87 and 0.77, respectively, for the training set were 0.86 and 0.72, respectively. The results indicated that the fitted model is robust, stable and satisfies all the prerequisites of the regression models. Combining intra-molecular descriptors with inter-molecular interaction descriptors between drug molecules and human serum albumin, the drug plasma protein binding could be modeled and predicted by the PPI-QSAR method successfully.

  17. Static headspace analysis of odorants in commercial rice proteins.

    PubMed

    Zhao, Jing; Boatright, William L

    2017-04-15

    Accurate identification of the odor-contributing compounds in aqueous slurries of rice proteins is necessary to improve their overall flavor characteristics. The objective of this study was to identify the primary odorants in rice protein slurries using static headspace analysis. Five commercial rice protein (RP) products, RP-G, RP-O, RP-RM, RP-RS1, and RP-RS2, were analyzed. RP-G contained the lowest levels of most of the odorants. Acetaldehyde was present in the highest amount in RP-O (0.434mg/m(3)). RP-RM had the highest levels of hexanal (5.907mg/m(3)), methanethiol (0.138mg/m(3)), pentanal (1.575mg/m(3)), and 2-pentylfuran (5.702mg/m(3)). Corresponding odor values were, 111, 86, 22 and 21, respectively. In RP-RS1 and RP-RS2, the predominant odorants were dimethyl disulfide, dimethyl trisulfide, and hexanal. The results showed the importance of the volatile compounds produced from amino acids, including the sulfur-containing compounds and acetaldehyde, as well as lipid oxidation derived odorants to the overall odor of rice proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Comparative analysis of ATRX, a chromatin remodeling protein.

    PubMed

    Park, Daniel J; Pask, Andrew J; Huynh, Kim; Renfree, Marilyn B; Harley, Vincent R; Graves, Jennifer A Marshall

    2004-09-15

    The ATRX protein, associated with X-linked alpha-thalassaemia, mental retardation and developmental abnormalities including genital dysgenesis, has been proposed to function as a global transcriptional regulator within a multi-protein complex. However, an understanding of the composition and mechanics of this machinery has remained elusive. We applied inter-specific comparative analysis to identify conserved elements which may be involved in regulating the conformation of chromatin. As part of this study, we cloned and sequenced the entire translatable coding region (7.4 kb) of the ATRX gene from a model marsupial (tammar wallaby, Macropus eugenii). We identify an ATRX ancestral core, conserved between plants, fish and mammals, comprising the cysteine-rich and SWI2/SNF2 helicase-like regions and protein interaction domains. Our data are consistent with the model of the cysteine-rich region as a DNA-binding zinc finger adjacent to a protein-binding (plant homeodomain-like) domain. Alignment of vertebrate ATRX sequences highlights other conserved elements, including a negatively charged mammalian sequence which we propose to be involved in binding of positively charged histone tails.

  19. Efficiency analysis of sampling protocols used in protein crystallization screening

    NASA Astrophysics Data System (ADS)

    Segelke, Brent W.

    2001-11-01

    In an effort to objectively compare the efficiency of protein crystallization screening techniques, a probability model of sampling efficiency is developed and used to calculate sampling efficiencies from experimental data. Three typical sampling protocols (grid screening, footprint screening, and random screening) are used to crystallize each of five proteins (Phospholipase A 2, Thaumatin, Catalase, Lysozyme, and Ribonuclease B). For each of the three sampling protocols, experiments are chosen from a large set of possible experiments generated by systematic combination of a number of parameters common in crystallization screens. Software has been developed to generate and select from the combinations with each of the three sampling protocols examined in this study. The protocols differ only in the order samples are chosen from the set of possible combinations. Random sampling is motivated by the "Incomplete Factorial" screen (Carter and Carter, J. Biol. Chem. 254 (1979) 12 219); sampling with subsets of four is motivated by the "Footprint" screen (Stura et al., J. Crystal Growth 122 (1992) 273) and sampling with subsets of twenty-four is motivated by the "Grid" screen (McPherson, Prepartion and Analysis of Protein Crystals, Wiley, New York, 1982). For the five proteins examined, random sampling has the greatest average efficiency. Additional benefits of random sampling are discussed.

  20. Domain tree-based analysis of protein architecture evolution.

    PubMed

    Forslund, Kristoffer; Henricson, Anna; Hollich, Volker; Sonnhammer, Erik L L

    2008-02-01

    Understanding the dynamics behind domain architecture evolution is of great importance to unravel the functions of proteins. Complex architectures have been created throughout evolution by rearrangement and duplication events. An interesting question is how many times a particular architecture has been created, a form of convergent evolution or domain architecture reinvention. Previous studies have approached this issue by comparing architectures found in different species. We wanted to achieve a finer-grained analysis by reconstructing protein architectures on complete domain trees. The prevalence of domain architecture reinvention in 96 genomes was investigated with a novel domain tree-based method that uses maximum parsimony for inferring ancestral protein architectures. Domain architectures were taken from Pfam. To ensure robustness, we applied the method to bootstrap trees and only considered results with strong statistical support. We detected multiple origins for 12.4% of the scored architectures. In a much smaller data set, the subset of completely domain-assigned proteins, the figure was 5.6%. These results indicate that domain architecture reinvention is a much more common phenomenon than previously thought. We also determined which domains are most frequent in multiply created architectures and assessed whether specific functions could be attributed to them. However, no strong functional bias was found in architectures with multiple origins.

  1. Analysis and Interpretation of Single Molecule Protein Unfolding Kinetics

    NASA Astrophysics Data System (ADS)

    Lannon, Herbert; Brujic, Jasna

    2012-02-01

    The kinetics of protein unfolding under a stretching force has been extensively studied by atomic force microscopy (AFM) over the past decade [1]. Experimental artifacts at the single molecule level introduce uncertainties in the data analysis that have led to several competing physical models for the unfolding process. For example, the unfolding dynamics of the protein ubiquitin under constant force has been described by probability distributions as diverse as exponential [2,3], a sum of exponentials, log-normal [4], and more recently a function describing static disorder in the Arrhenius model [5]. A new method for data analysis is presented that utilizes maximum likelihood estimation (MLE) combined with other traditional statistical tests to unambiguously rank the consistency of these and other models with the experimental data. These techniques applied to the ubiquitin unfolding data shows that the probability of unfolding is best fit with a stretched exponential distribution, with important implications on the complexity of the mechanism of protein unfolding. [4pt] [1] Carrion-Vazquez, et. al. Springer Series in Biophys. 2006 [0pt] [2] Fernandez et. al. Science 2004 [0pt] [3] Brujic et. al. Nat. Phys 2006 [0pt] [4] Garcia-Manyes et. al. Biophys. J. 2007 [0pt] [5] Kuo et. al. PNAS 2010

  2. Quantiprot - a Python package for quantitative analysis of protein sequences.

    PubMed

    Konopka, Bogumił M; Marciniak, Marta; Dyrka, Witold

    2017-07-17

    The field of protein sequence analysis is dominated by tools rooted in substitution matrices and alignments. A complementary approach is provided by methods of quantitative characterization. A major advantage of the approach is that quantitative properties defines a multidimensional solution space, where sequences can be related to each other and differences can be meaningfully interpreted. Quantiprot is a software package in Python, which provides a simple and consistent interface to multiple methods for quantitative characterization of protein sequences. The package can be used to calculate dozens of characteristics directly from sequences or using physico-chemical properties of amino acids. Besides basic measures, Quantiprot performs quantitative analysis of recurrence and determinism in the sequence, calculates distribution of n-grams and computes the Zipf's law coefficient. We propose three main fields of application of the Quantiprot package. First, quantitative characteristics can be used in alignment-free similarity searches, and in clustering of large and/or divergent sequence sets. Second, a feature space defined by quantitative properties can be used in comparative studies of protein families and organisms. Third, the feature space can be used for evaluating generative models, where large number of sequences generated by the model can be compared to actually observed sequences.

  3. Analysis of Protein O-GlcNAcylation by Mass Spectrometry.

    PubMed

    Ma, Junfeng; Hart, Gerald W

    2017-02-02

    O-linked β-D-N-acetyl glucosamine (O-GlcNAc) addition (O-GlcNAcylation), a post-translational modification of serine/threonine residues of proteins, is involved in diverse cellular metabolic and signaling pathways. Aberrant O-GlcNAcylation underlies the initiation and progression of multiple chronic diseases including diabetes, cancer, and neurodegenerative diseases. Numerous methods have been developed for the analysis of protein O-GlcNAcylation, but instead of discussing the classical biochemical techniques, this unit covers O-GlcNAc characterization by combining several enrichment methods and mass spectrometry detection techniques [including collision-induced dissociation (CID), higher energy collision dissociation (HCD), and electron transfer dissociation (ETD) mass spectrometry]. © 2017 by John Wiley & Sons, Inc.

  4. Towards proteomic analysis of milk proteins in historical building materials

    NASA Astrophysics Data System (ADS)

    Kuckova, S.; Crhova, M.; Vankova, L.; Hnizda, A.; Hynek, R.; Kodicek, M.

    2009-07-01

    The addition of proteinaceous binders to mortars and plasters has a long tradition. The protein additions were identified in many sacral and secular historical buildings. For this method of peptide mass mapping, three model mortar samples with protein additives were prepared. These samples were analysed fresh (1-2 weeks old) and after 9 months of natural ageing. The optimal duration of tryptic cleavage (2 h) and the lowest amount of material needed for relevant analysis of fresh and weathered samples were found; the sufficient amounts of weathered and fresh mortars were set to 0.05 and 0.005 g. The list of main tryptic peptides coming from milk additives (bovine milk, curd, and whey), their relative intensities and theoretical amino acid sequences assignment is presented. Several sequences have been "de novo" confirmed by mass spectrometry.

  5. [Molecularly imprinted polymers in electro analysis of proteins].

    PubMed

    Shumyantseva, V V; Bulko, T V; Baychorov, I Kh; Archakov, A I

    2015-01-01

    In the review the main approaches to creation of recognition materials capable of competing with biological specific receptors, (polymeric analogs of antibodies or molecularly imprinted polymers, MIP) for the electro analysis of functionally significant proteins such as a myoglobin, troponin T, albumin, human ferritin, calmodulin are considered. The main types of monomers for MIP fabrication, and methods for MIP/protein interactions, such as a surface plasmon resonance (SPR), nanogravimetry with use of the quartz crystal resonator (QCM), spectral and electrochemical methods are discussed. Experimental data on electrochemical registration of a myoglobin using MIP/electrode are presented. For a development of electrochemical sensor systems based on MIPs, o-phenylenediamine (1,2-diaminobenzene was used as a monomer. It was shown that the imprinting factor Imax(MIP)/Imax(NIP), calculated as a myoglobin signal ratio when embedding in MIP to a myoglobin signal when embedding in the polymer received without molecular template (NIP) corresponds 2-4.

  6. Hyperdimensional Analysis of Amino Acid Pair Distributions in Proteins

    PubMed Central

    Henriksen, Svend B.; Arnason, Omar; Söring, Jón; Petersen, Steffen B.

    2011-01-01

    Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis. PMID:22174733

  7. Hydrophobic-cluster analysis of plant protein sequences. A domain homology between storage and lipid-transfer proteins.

    PubMed Central

    Henrissat, B; Popineau, Y; Kader, J C

    1988-01-01

    Hydrophobic-cluster analysis was used to characterize a conserved domain located near the C-terminal amino acid sequence of wheat (Triticum aestivum) storage proteins. This domain was transformed into a linear template for a global search for similarities in over 5200 protein sequences. In addition to proteins that had already been found to exhibit homology to wheat storage proteins, a previously unreported homology was found with non-specific lipid-transfer proteins from castor bean (Ricinus communis) and from spinach (Spinacia oleracea) leaf. Hydrophobic-cluster analysis of various members of the present protein group clearly shows a typical domain structure where (i) variable and conserved domains are located along the sequence at precise positions, (ii) the conserved domains probably reflect a common ancestor, and (iii) the unique properties of a given protein (chain cut into subunits, repetitive domains, trypsin-inhibitor active site) are associated with the variable domains. PMID:3214430

  8. Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

    PubMed Central

    Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

    2016-01-01

    We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

  9. Analytical methods in DNA and protein adduct analysis.

    PubMed

    Koivisto, Pertti; Peltonen, Kimmo

    2010-11-01

    DNA or protein adducts are reaction products of endogenous or exogenous chemicals and cellular macromolecules. Adducts are useful in toxicological studies and/or human biomonitoring exercises. In particular, DNA damage provides invaluable information for risk analysis. Second, metabolites or conjugates can be regarded as markers of phase II reactions though they may not give accurate information about the levels of reactive and damage-provoking reactive compounds or intermediates. Electrophiles are often short-lived molecules and therefore difficult to monitor. In contrast, adducts are often chemically stable, though their levels in biological samples are low, which makes their detection challenging. The assay of adducts is similar to the analysis of any other trace organic molecule, i.e. problems with the matrix and small amounts of analytes in samples. The (32)P-postlabelling assay is a specific method for DNA adducts but immunochemical and fluorescence-based methods have been developed which can detect adducts linked to both DNA and protein. Tandem mass spectrometry, particularly if combined with ultrahigh-performance liquid chromatography, is currently the recommended detection technique; however investigators are striving to develop novel ways to achieve greater sensitivity. Standards are a prerequisite in adduct analysis, but unfortunately they are seldom commercially available.

  10. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  11. Mutational analysis of the ribosomal protein Rpl10 from yeast.

    PubMed

    Hofer, Anne; Bussiere, Cyril; Johnson, Arlen W

    2007-11-09

    Yeast Rpl10 belongs to the L10e family of ribosomal proteins. In the large (60 S) subunit, Rpl10 is positioned in a cleft between the central protuberance and the GTPase-activating center. It is loaded into the 60 S subunit at a late step in maturation. We have shown previously that Rpl10 is required for the release of the Crm1-dependent nuclear export adapter Nmd3, an event that also requires the cytoplasmic GTPase Lsg1. Here we have carried out an extensive mutational analysis of Rpl10 to identify mutations that would allow us to map activities to distinct domains of the protein to begin to understand the molecular interaction between Rpl10 and Nmd3. We found that mutations in a central loop (amino acids 102-112) had a significant impact on the release of Nmd3. This loop is unstructured in the crystal and solution structures of prokaryotic Rpl10 orthologs. Thus, the loop is not necessary for stable interaction of Rpl10 with the ribosome, suggesting that it plays a dynamic role in ribosome function or regulating the association of other factors. We also found that mutant Rpl10 proteins were engineered to be unable to bind to the ribosome accumulated in the nucleus. This was unexpected and may suggest a nuclear role for Rpl10.

  12. Integrated visual analysis of protein structures, sequences, and feature data

    PubMed Central

    2015-01-01

    Background To understand the molecular mechanisms that give rise to a protein's function, biologists often need to (i) find and access all related atomic-resolution 3D structures, and (ii) map sequence-based features (e.g., domains, single-nucleotide polymorphisms, post-translational modifications) onto these structures. Results To streamline these processes we recently developed Aquaria, a resource offering unprecedented access to protein structure information based on an all-against-all comparison of SwissProt and PDB sequences. In this work, we provide a requirements analysis for several frequently occuring tasks in molecular biology and describe how design choices in Aquaria meet these requirements. Finally, we show how the interface can be used to explore features of a protein and gain biologically meaningful insights in two case studies conducted by domain experts. Conclusions The user interface design of Aquaria enables biologists to gain unprecedented access to molecular structures and simplifies the generation of insight. The tasks involved in mapping sequence features onto structures can be conducted easier and faster using Aquaria. PMID:26329268

  13. Laminated microfluidic system for small sample protein analysis

    PubMed Central

    Saedinia, Sara; Nastiuk, Kent L.; Krolewski, John J.; Li, G. P.; Bachman, Mark

    2014-01-01

    We describe a technology based on lamination that allows for the production of highly integrated 3D devices suitable for performing a wide variety of microfluidic assays. This approach uses a suite of microfluidic coupons (“microfloupons”) that are intended to be stacked as needed to produce an assay of interest. Microfloupons may be manufactured in paper, plastic, gels, or other materials, in advance, by different manufacturers, then assembled by the assay designer as needed. To demonstrate this approach, we designed, assembled, and characterized a microfloupon device that performs sodium-dodecyl-sulfate polyacrylamide gel electrophoresis on a small sample of protein. This device allowed for the manipulation and transport of small amounts of protein sample, tight injection into a thin polyacrylamide gel, electrophoretic separation of the proteins into bands, and subsequent removal of the gel from the device for imaging and further analysis. The microfloupons are rugged enough to handle and can be easily aligned and laminated, allowing for a variety of different assays to be designed and configured by selecting appropriate microfloupons. This approach provides a convenient way to perform assays that have multiple steps, relieving the need to design highly sophisticated devices that incorporate all functions in a single unit, while still achieving the benefits of small sample size, automation, and high speed operation. PMID:24753728

  14. Analysis of antifreeze protein activity using colorimetric gold nanosensors

    NASA Astrophysics Data System (ADS)

    Jing, Xu; Choi, Ho-seok; Park, Ji-In; Kim, Young-Pil

    2015-07-01

    High activity and long stability of antifreeze proteins (AFPs), also known as ice-binding proteins (IBPs), are necessary for exerting their physiological functions in biotechnology and cryomedicine. Here we report a simple analysis of antifreeze protein activity and stability based on self-assembly of gold nanoparticles (AuNPs) via freezing and thawing cycles. While the mercaptosuccinic acid-capped AuNP (MSA-AuNP) was easily self-assembled after a freezing/thawing cycle, due to the mechanical attack of ice crystal on the MSA-AuNP surface, the presence of AFP impeded the self-assembly of MSA-AuNP via the interaction of AFP with ice crystals via freezing and thawing cycles, which led to a strong color in the MSA-AuNP solution. As a result, the aggregation parameter (E520/E650) of MSA-AuNP showed the rapid detection of both activity and stability of AFPs. We suggest that our newly developed method is very suitable for measuring antifreeze activity and stability in a simple and rapid manner with reliable quantification.

  15. Molecular analysis of the muscle protein projectin in Lepidoptera.

    PubMed

    Ayme-Southgate, A J; Turner, L; Southgate, R J

    2013-01-01

    Striated muscles of both vertebrates and insects contain a third filament composed of the giant proteins, namely kettin and projectin (insects) and titin (vertebrates). All three proteins have been shown to contain several domains implicated in conferring elasticity, in particular a PEVK segment. In this study, the characterization of the projectin protein in the silkmoth, Bombyx mori L. (Lepidoptera: Bombycidae), and the monarch butterfly, Danaus plexippus L. (Lepidoptera: Nymphalidae), as well as a partial characterization in the Carolina sphinx, Manduca sexta L. (Lepidoptera: Sphingidae), are presented. This study showed that, similar to other insects, projectin's overall modular organization was conserved, but in contrast, the PEVK region had a highly divergent sequence. The analysis of alternative splicing in the PEVK region revealed a small number of possible isoforms and the lack of a flight-muscle specific variant, both characteristics being in sharp contrast with findings from other insects. The possible correlation with difference in flight muscle stiffness and physiology between Lepidoptera and other insect orders is discussed.

  16. Heat capacity of solid proteins by thermal analysis

    SciTech Connect

    Zhang, Ge; Wunderlich, B.

    1997-11-01

    In a continuing effort to better understand the thermodynamic properties of proteins, solid state heat capacities of poly(amino acid)s of all 21 naturally occurring amino 4 copoly(amino acid)s and about 10 proteins have been analyzed by now using the Advanced Thermal Analysis System, ATHAS. The experimental measurements were performed with adiabatic and differential scanning calorimetry from 10 to about 450 K. The heat capacities of the samples in their pure, solid states are linked to an approximate vibrational spectrum by making use of known group vibrations and a set of parameters, {Theta}{sub 1} and {Theta}{sub 3}, of the Tarasov function for the skeletal vibrations. Good agreement is found between experiment and calculation with root mean square errors mostly within {+-}3%. The experimental data were analyzed also with an empirical addition scheme using data for the poly(amino acid)s. Based on this study, vibrational heat capacity can now be predicted for all proteins with an accuracy comparable to common experiments. Furthermore, gradual transitions, indicative of molecular motion prior to devitrification, melting, or decomposition, can be identified. The new experimental data compared here with the prior samples are: bovine {beta}-lactoglobulin, chicken lysozyme and ovalbumin.

  17. Pathway Analysis Incorporating Protein-Protein Interaction Networks Identified Candidate Pathways for the Seven Common Diseases

    PubMed Central

    Lin, Peng-Lin; Yu, Ya-Wen

    2016-01-01

    Pathway analysis has become popular as a secondary analysis strategy for genome-wide association studies (GWAS). Most of the current pathway analysis methods aggregate signals from the main effects of single nucleotide polymorphisms (SNPs) in genes within a pathway without considering the effects of gene-gene interactions. However, gene-gene interactions can also have critical effects on complex diseases. Protein-protein interaction (PPI) networks have been used to define gene pairs for the gene-gene interaction tests. Incorporating the PPI information to define gene pairs for interaction tests within pathways can increase the power for pathway-based association tests. We propose a pathway association test, which aggregates the interaction signals in PPI networks within a pathway, for GWAS with case-control samples. Gene size is properly considered in the test so that genes do not contribute more to the test statistic simply due to their size. Simulation studies were performed to verify that the method is a valid test and can have more power than other pathway association tests in the presence of gene-gene interactions within a pathway under different scenarios. We applied the test to the Wellcome Trust Case Control Consortium GWAS datasets for seven common diseases. The most significant pathway is the chaperones modulate interferon signaling pathway for Crohn’s disease (p-value = 0.0003). The pathway modulates interferon gamma, which induces the JAK/STAT pathway that is involved in Crohn’s disease. Several other pathways that have functional implications for the seven diseases were also identified. The proposed test based on gene-gene interaction signals in PPI networks can be used as a complementary tool to the current existing pathway analysis methods focusing on main effects of genes. An efficient software implementing the method is freely available at http://puppi.sourceforge.net. PMID:27622767

  18. [A Method for Protein Photo-cross-linking in Living Cells Facilitating Analysis of Physiological Interactions of Proteins].

    PubMed

    Hino, Nobumasa

    2015-01-01

    In living cells, most proteins form complexes with other proteins to exert their functions. Since protein functions are regulated in response to changes in the cellular environment, the components of the complexes can vary; therefore, proteins often interact in a weak and transient manner. To capture such labile protein interactions, we have developed a method for photo-cross-linking of proteins directly interacting in mammalian cells; this method involves expansion of the genetic code and site-specific incorporation of photoreactive amino acids into proteins. Upon cross-linking, protein complexes are stabilized by a covalent bond and can be readily isolated from cell extracts without the problems usually associated with simple affinity purification methods such as co-immunoprecipitation. Photo-cross-linkers have another benefit: they react exclusively with molecules within a range defined by the linker length. This property becomes useful for determining the binding interface of two proteins because the linkers can be introduced in a site-directed manner with our method. In this review, we first describe the expansion of the genetic code of mammalian cells for the incorporation of non-natural amino acids into proteins. Then, we introduce our recent applications and developments of the cross-linking method: identification of intracellular binding partners of the signaling protein growth factor receptor binding protein 2; analysis of the binding between membrane proteins on the cell surface; and a novel photoreactive amino acid that enables wide-ranging photo-cross-linking.

  19. Photonic Crystal Hydrogel Enhanced Plasmonic Staining for Multiplexed Protein Analysis.

    PubMed

    Mu, Zhongde; Zhao, Xiangwei; Huang, Yin; Lu, Meng; Gu, Zhongze

    2015-12-02

    Plasmonic nanoparticles are commonly used as optical transducers in sensing applications. The optical signals resulting from the interaction of analytes and plamsonic nanoparticles are influenced by surrounding physical structures where the nanoparticles are located. This paper proposes inverse opal photonic crystal hydrogel as 3D structure to improve Raman signals from plasmonic staining. By hybridization of the plasmonic nanoparticles and photonic crystal, surface-enhanced Raman spectroscopy (SERS) analysis of multiplexed protein is realized. It benefits the Raman analysis by providing high-density "hot spots" in 3D and extra enhancement of local electromagnetic field at the band edge of PhC with periodic refractive index distribution. The strong interaction of light and the hybrid 3D nanostructure offers new insights into plasmonic nanoparticle applications and biosensor design.

  20. Comparative analysis and assessment of M. tuberculosis H37Rv protein-protein interaction datasets

    PubMed Central

    2011-01-01

    predicted physical interologs from IntAct and S. aureus MRSA252 pull-down PPIs. Comparative analysis with several representative two-hybrid PPI datasets in other species further confirms that the H37Rv B2H PPI dataset is of low quality. Next, to test the possibility that the H37Rv STRING PPIs are not purely direct physical interactions, we compare M. tuberculosis H37Rv protein pairs that catalyze adjacent steps in enzymatic reactions to B2H PPIs and predicted PPIs in STRING, which shows it has much lower similarities with the B2H PPIs than with STRING PPIs. This result strongly suggests that the H37Rv STRING PPIs more likely correspond to indirect relationships between protein pairs than to B2H PPIs. For more precise support, we turn to S. cerevisiae for its comprehensively studied interactome. We compare S. cerevisiae predicted PPIs in STRING to three independent protein relationship datasets which respectively comprise PPIs reported in Y2H assays, protein pairs reported to be in the same protein complexes, and protein pairs that catalyze successive reaction steps in enzymatic reactions. Our analysis reveals that S. cerevisiae predicted STRING PPIs have much higher similarity to the latter two types of protein pairs than to two-hybrid PPIs. As H37Rv STRING PPIs are predicted using similar methods as S. cerevisiae predicted STRING PPIs, this suggests that these H37Rv STRING PPIs are more likely to correspond to the latter two types of protein pairs rather than to two-hybrid PPIs as well. Conclusions The H37Rv B2H PPI dataset has low quality. It should not be used as the gold standard to assess the quality of other (possibly predicted) H37Rv PPI datasets. The H37Rv STRING PPI dataset also has low quality; nevertheless, a subset consisting of STRING PPIs with score ≥770 has satisfactory quality. However, these STRING “PPIs” should be interpreted as functional associations, which include a substantial portion of indirect protein interactions, rather than direct physical

  1. Analysis of zinc binding sites in protein crystal structures.

    PubMed Central

    Alberts, I. L.; Nadassy, K.; Wodak, S. J.

    1998-01-01

    The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations. PMID:10082367

  2. Prioritizing Disease Candidate Proteins in Cardiomyopathy-Specific Protein-Protein Interaction Networks Based on “Guilt by Association” Analysis

    PubMed Central

    He, Weiming; Li, Weiguo; Qu, Xiaoli; Liang, Binhua; Gao, Qianping; Feng, Chenchen; Jia, Xu; Lv, Yana; Zhang, Siya; Li, Xia

    2013-01-01

    The cardiomyopathies are a group of heart muscle diseases which can be inherited (familial). Identifying potential disease-related proteins is important to understand mechanisms of cardiomyopathies. Experimental identification of cardiomyophthies is costly and labour-intensive. In contrast, bioinformatics approach has a competitive advantage over experimental method. Based on “guilt by association” analysis, we prioritized candidate proteins involving in human cardiomyopathies. We first built weighted human cardiomyopathy-specific protein-protein interaction networks for three subtypes of cardiomyopathies using the known disease proteins from Online Mendelian Inheritance in Man as seeds. We then developed a method in prioritizing disease candidate proteins to rank candidate proteins in the network based on “guilt by association” analysis. It was found that most candidate proteins with high scores shared disease-related pathways with disease seed proteins. These top ranked candidate proteins were related with the corresponding disease subtypes, and were potential disease-related proteins. Cross-validation and comparison with other methods indicated that our approach could be used for the identification of potentially novel disease proteins, which may provide insights into cardiomyopathy-related mechanisms in a more comprehensive and integrated way. PMID:23940716

  3. Analysis of coevolution in nonstructural proteins of chikungunya virus.

    PubMed

    Jain, Jaspreet; Mathur, Kalika; Shrinet, Jatin; Bhatnagar, Raj K; Sunil, Sujatha

    2016-06-02

    RNA viruses are characterized by high rate of mutations mainly due to the lack of proofreading repair activities associated with its RNA-dependent RNA-polymerase (RdRp). In case of arboviruses, this phenomenon has lead to the existence of mixed population of genomic variants within the host called quasi-species. The stability of strains within the quasi-species lies on mutations that are positively selected which in turn depend on whether these mutations are beneficial in either or both hosts. Coevolution of amino acids (aa) is one phenomenon that leads to establishment of favorable traits in viruses and leading to their fitness. Fourteen CHIKV clinical samples collected over three years were subjected to RT-PCR, the four non-structural genes amplified and subjected to various genetic analyses. Coevolution analysis showed 30 aa pairs coevolving in nsP1, 23 aa pairs coevolving in nsP2, 239 in nsP3 and 46 aa coevolving pairs in nsP4 when each non-structural protein was considered independently. Further analysis showed that 705 amino acids pairs of the non-structural polyproteins coevolved together with a correlation coefficient of ≥0.5. Functional relevance of these coevolving amino acids in all the nonstructural proteins of CHIKV were predicted using Eukaryotic Linear Motifs (ELMs) of human. The present study was undertaken to study co-evolving amino acids in the non-structural proteins of chikungunya virus (CHIKV), an important arbovirus. It was observed that several amino acids residues were coevolving and shared common functions.

  4. Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes.

    PubMed

    Krause, Frank

    2006-07-01

    It is an essential and challenging task to unravel protein-protein interactions in their actual in vivo context. Native gel systems provide a separation platform allowing the analysis of protein complexes on a rather proteome-wide scale in a single experiment. This review focus on blue-native (BN)-PAGE as the most versatile and successful gel-based approach to separate soluble and membrane protein complexes of intricate protein mixtures derived from all biological sources. BN-PAGE is a charge-shift method with a running pH of 7.5 relying on the gentle binding of anionic CBB dye to all membrane and many soluble protein complexes, leading to separation of protein species essentially according to their size and superior resolution than other fractionation techniques can offer. The closely related colorless-native (CN)-PAGE, whose applicability is restricted to protein species with intrinsic negative net charge, proved to provide an especially mild separation capable of preserving weak protein-protein interactions better than BN-PAGE. The essential conditions determining the success of detecting protein-protein interactions are the sample preparations, e.g. the efficiency/mildness of the detergent solubilization of membrane protein complexes. A broad overview about the achievements of BN- and CN-PAGE studies to elucidate protein-protein interactions in organelles and prokaryotes is presented, e.g. the mitochondrial protein import machinery and oxidative phosphorylation supercomplexes. In many cases, solubilization with digitonin was demonstrated to facilitate an efficient and particularly gentle extraction of membrane protein complexes prone to dissociation by treatment with other detergents. In general, analyses of protein interactomes should be carried out by both BN- and CN-PAGE.

  5. Molecular Analysis of the Acinetobacter baumannii Biofilm-Associated Protein

    PubMed Central

    Goh, H. M. Sharon; Beatson, Scott A.; Totsika, Makrina; Moriel, Danilo G.; Phan, Minh-Duy; Szubert, Jan; Runnegar, Naomi; Sidjabat, Hanna E.; Paterson, David L.; Nimmo, Graeme R.; Lipman, Jeffrey

    2013-01-01

    Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that BapMS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bapMS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates. PMID:23956398

  6. Proteomic analysis of membrane proteins of vero cells: exploration of potential proteins responsible for virus entry.

    PubMed

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong; Sun, Dongbo

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells.

  7. Flow Cytometric Single-Cell Analysis for Quantitative in Vivo Detection of Protein-Protein Interactions via Relative Reporter Protein Expression Measurement.

    PubMed

    Wu, Lina; Wang, Xu; Zhang, Jianqiang; Luan, Tian; Bouveret, Emmanuelle; Yan, Xiaomei

    2017-03-07

    Cell-based two-hybrid assays have been key players in identifying pairwise interactions, yet quantitative measurement of protein-protein interactions in vivo remains challenging. Here, we show that by using relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, quantitative analysis of protein interactions in a bacterial adenylate cyclase two-hybrid (BACTH) system can be achieved. A multicolor flow cytometer was used to measure simultaneously the expression levels of one of the two putative interacting proteins and the β-galactosidase (β-gal) reporter protein upon dual immunofluorescence staining. Single-cell analysis revealed that there exists bistability in the BACTH system and the RRPE is an intrinsic characteristic associated with the binding strength between the two interacting proteins. The RRPE-BACTH method provides an efficient tool to confirm interacting pairs of proteins, investigate determinant residues in protein-protein interaction, and compare interaction strength of different pairs.

  8. An Introductory Classroom Exercise on Protein Molecular Model Visualization and Detailed Analysis of Protein-Ligand Binding

    ERIC Educational Resources Information Center

    Poeylaut-Palena, Andres, A.; de los Angeles Laborde, Maria

    2013-01-01

    A learning module for molecular level analysis of protein structure and ligand/drug interaction through the visualization of X-ray diffraction is presented. Using DeepView as molecular model visualization software, students learn about the general concepts of protein structure. This Biochemistry classroom exercise is designed to be carried out by…

  9. An Introductory Classroom Exercise on Protein Molecular Model Visualization and Detailed Analysis of Protein-Ligand Binding

    ERIC Educational Resources Information Center

    Poeylaut-Palena, Andres, A.; de los Angeles Laborde, Maria

    2013-01-01

    A learning module for molecular level analysis of protein structure and ligand/drug interaction through the visualization of X-ray diffraction is presented. Using DeepView as molecular model visualization software, students learn about the general concepts of protein structure. This Biochemistry classroom exercise is designed to be carried out by…

  10. Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

    PubMed

    Reiz, Bela; Li, Liang

    2010-09-01

    Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.

  11. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    PubMed Central

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  12. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  13. Techniques for the Analysis of Protein-Protein Interactions in Vivo1[OPEN

    PubMed Central

    Xing, Shuping; Wallmeroth, Niklas; Berendzen, Kenneth W.

    2016-01-01

    Identifying key players and their interactions is fundamental for understanding biochemical mechanisms at the molecular level. The ever-increasing number of alternative ways to detect protein-protein interactions (PPIs) speaks volumes about the creativity of scientists in hunting for the optimal technique. PPIs derived from single experiments or high-throughput screens enable the decoding of binary interactions, the building of large-scale interaction maps of single organisms, and the establishment of cross-species networks. This review provides a historical view of the development of PPI technology over the past three decades, particularly focusing on in vivo PPI techniques that are inexpensive to perform and/or easy to implement in a state-of-the-art molecular biology laboratory. Special emphasis is given to their feasibility and application for plant biology as well as recent improvements or additions to these established techniques. The biology behind each method and its advantages and disadvantages are discussed in detail, as are the design, execution, and evaluation of PPI analysis. We also aim to raise awareness about the technological considerations and the inherent flaws of these methods, which may have an impact on the biological interpretation of PPIs. Ultimately, we hope this review serves as a useful reference when choosing the most suitable PPI technique. PMID:27208310

  14. Affinity purification-mass spectrometry and network analysis to understand protein-protein interactions.

    PubMed

    Morris, John H; Knudsen, Giselle M; Verschueren, Erik; Johnson, Jeffrey R; Cimermancic, Peter; Greninger, Alexander L; Pico, Alexander R

    2014-11-01

    By determining protein-protein interactions in normal, diseased and infected cells, we can improve our understanding of cellular systems and their reaction to various perturbations. In this protocol, we discuss how to use data obtained in affinity purification-mass spectrometry (AP-MS) experiments to generate meaningful interaction networks and effective figures. We begin with an overview of common epitope tagging, expression and AP practices, followed by liquid chromatography-MS (LC-MS) data collection. We then provide a detailed procedure covering a pipeline approach to (i) pre-processing the data by filtering against contaminant lists such as the Contaminant Repository for Affinity Purification (CRAPome) and normalization using the spectral index (SIN) or normalized spectral abundance factor (NSAF); (ii) scoring via methods such as MiST, SAInt and CompPASS; and (iii) testing the resulting scores. Data formats familiar to MS practitioners are then transformed to those most useful for network-based analyses. The protocol also explores methods available in Cytoscape to visualize and analyze these types of interaction data. The scoring pipeline can take anywhere from 1 d to 1 week, depending on one's familiarity with the tools and data peculiarities. Similarly, the network analysis and visualization protocol in Cytoscape takes 2-4 h to complete with the provided sample data, but we recommend taking days or even weeks to explore one's data and find the right questions.

  15. Analysis of protein-protein interaction network in chronic obstructive pulmonary disease.

    PubMed

    Yuan, Y P; Shi, Y H; Gu, W C

    2014-10-31

    Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality throughout the world. The purpose of our study was to uncover biomarkers and explore its pathogenic mechanisms at the molecular level. The gene expression profiles of COPD samples and normal controls were downloaded from Gene Expression Omnibus. Matlab was used for data preprocessing and SAM4.0 was applied to determine the differentially expressed genes (DEGs). Furthermore, a protein-protein interaction (PPI) network was constructed by mapping the DEGs into PPI data, and functional analysis of the network was conducted with BiNGO. A total of 348 DEGs and 765 interactive genes were identified. The hub genes were mainly involved in metabolic processes and ribosome biogenesis. Several genes related to COPD in the PPI network were found, including CAMK1D, ALB, KIT, and DDX3Y. In conclusion, CAMK1D, ALB, KIT, and DDX3Y were chosen as candidate genes, which have the potential to be biomarkers or candidate target molecules to apply in clinical diagnosis and treatment of COPD.

  16. Evolutionary analysis and interaction prediction for protein-protein interaction network in geometric space.

    PubMed

    Huang, Lei; Liao, Li; Wu, Cathy H

    2017-01-01

    Prediction of protein-protein interaction (PPI) remains a central task in systems biology. With more PPIs identified, forming PPI networks, it has become feasible and also imperative to study PPIs at the network level, such as evolutionary analysis of the networks, for better understanding of PPI networks and for more accurate prediction of pairwise PPIs by leveraging the information gained at the network level. In this work we developed a novel method that enables us to incorporate evolutionary information into geometric space to improve PPI prediction, which in turn can be used to select and evaluate various evolutionary models. The method is tested with cross-validation using human PPI network and yeast PPI network data. The results show that the accuracy of PPI prediction measured by ROC score is increased by up to 14.6%, as compared to a baseline without using evolutionary information. The results also indicate that our modified evolutionary model DANEOsf-combining a gene duplication/neofunctionalization model and scale-free model-has a better fitness and prediction efficacy for these two PPI networks. The improved PPI prediction performance may suggest that our DANEOsf evolutionary model can uncover the underlying evolutionary mechanism for these two PPI networks better than other tested models. Consequently, of particular importance is that our method offers an effective way to select evolutionary models that best capture the underlying evolutionary mechanisms, evaluating the fitness of evolutionary models from the perspective of PPI prediction on real PPI networks.

  17. Mutation analysis of barley malt protein Z4 and protein Z7 on beer foam stability.

    PubMed

    Iimure, Takashi; Kimura, Tatsuji; Araki, Shigeki; Kihara, Makoto; Sato, Masahide; Yamada, Shinji; Shigyou, Tatsuro; Sato, Kazuhiro

    2012-02-15

    Beer foam stability is an important characteristic. It has been suggested that isoforms of protein Z, that is, protein Z4 and protein Z7, contribute to beer foam stability. We investigated the relationship between beer foam stability and protein Z4 and protein Z7 using their deficient mutants. As a protein Z4-deficient mutant, cv. Pirkka was used. Protein Z7 deficiency was screened in 1564 barley accessions in the world collection of Okayama University, Japan. The barley samples from normal, protein Z4-deficient, protein Z7-deficient, and double-deficient were genotyped in F(2) populations and then pooled based on the DNA marker genotypes of protein Z4 and protein Z7. For a brewing trial, F(5) pooled subpopulations were used. After malting and brewing, the foam stability was determined, and the results showed that the levels of foam stability in the four samples were comparable. Two-dimensional gel electrophoresis was used to investigate the proteome in these beer samples. The results showed that low molecular weight proteins, including lipid transfer protein (LTP2), in the deficient mutants were higher than those in the normal sample. Our results suggest that the contribution of protein Z4 and protein Z7 to beer foam stability was not greater than that of other beer proteins.

  18. Category Theoretic Analysis of Hierarchical Protein Materials and Social Networks

    PubMed Central

    Spivak, David I.; Giesa, Tristan; Wood, Elizabeth; Buehler, Markus J.

    2011-01-01

    Materials in biology span all the scales from Angstroms to meters and typically consist of complex hierarchical assemblies of simple building blocks. Here we describe an application of category theory to describe structural and resulting functional properties of biological protein materials by developing so-called ologs. An olog is like a “concept web” or “semantic network” except that it follows a rigorous mathematical formulation based on category theory. This key difference ensures that an olog is unambiguous, highly adaptable to evolution and change, and suitable for sharing concepts with other olog. We consider simple cases of beta-helical and amyloid-like protein filaments subjected to axial extension and develop an olog representation of their structural and resulting mechanical properties. We also construct a representation of a social network in which people send text-messages to their nearest neighbors and act as a team to perform a task. We show that the olog for the protein and the olog for the social network feature identical category-theoretic representations, and we proceed to precisely explicate the analogy or isomorphism between them. The examples presented here demonstrate that the intrinsic nature of a complex system, which in particular includes a precise relationship between structure and function at different hierarchical levels, can be effectively represented by an olog. This, in turn, allows for comparative studies between disparate materials or fields of application, and results in novel approaches to derive functionality in the design of de novo hierarchical systems. We discuss opportunities and challenges associated with the description of complex biological materials by using ologs as a powerful tool for analysis and design in the context of materiomics, and we present the potential impact of this approach for engineering, life sciences, and medicine. PMID:21931622

  19. Genetic analysis of a Drosophila microtubule-associated protein

    PubMed Central

    1992-01-01

    The 205-kD microtubule-associated protein (205K MAP) is one of the principal MAPs in Drosophila. 205K MAP is similar to the HeLa 210K/MAP4 family of MAPs since it shares the following biochemical properties: it is present in several isoforms, has a molecular mass of approximately 200 kD, and is thermostable. Furthermore, immuno-crossreactivity has been observed between mouse MAP4, HeLa 210K, and Drosophila 205K MAP. Currently, there is little information concerning the biological function of this group of nonmotor MAPs. We have used a classical genetic approach to try to identify the role of the 205K MAP in Drosophila by isolating mutations in the 205K MAP gene. An F2 lethal screen was used to acquire deficiencies of 100EF, the chromosomal location of the 205K MAP gene. Drosophila bearing a homozygous deficiency for the 205K MAP region are fully viable and show no obvious phenotype. A recently developed polymerase chain reaction screen was also used to recover five P-element insertions upstream from the 205K MAP gene. Western blot analysis has shown that these insertions result in hypomorphic mutations of the 205K MAP gene. As was seen with animals that have no 205K MAP, these mutations appear to have no phenotype. These data unambiguously demonstrate that the 205K MAP gene is inessential for development. These results also suggest that there may exist protein(s) with redundant function that can substitute for 205K MAP. PMID:1309812

  20. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity.

    PubMed

    Watson, Eleanor; Sherry, Aileen; Inglis, Neil F; Lainson, Alex; Jyothi, Dushyanth; Yaga, Raja; Manson, Erin; Imrie, Lisa; Everest, Paul; Smith, David G E

    2014-09-01

    Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC-ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith-Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  1. Sequence analysis and phylogenetic study of some toxin proteins of snakes and related non-toxin proteins of chordates.

    PubMed

    Panda, Subhamay; Chandra, Goutam

    2013-01-01

    Snakes are equipped with their venomic armory to tackle different prey and predators in adverse natural world. The venomic composition of snakes is a mix of biologically active proteins and polypeptides. Among different components snake venom cytotoxins and short neurotoxin are non-enzymatic polypeptide candidates with in the venom. These two components structurally resembled to three-finger protein superfamily specific scaffold. Different non-toxin family members of three-finger protein superfamily are involved in different biological roles. In the present study we analyzed the snake venom cytotoxins, short neurotoxins and related non-toxin proteins of different chordates in terms of amino acid sequence level diversification profile, polarity profile of amino acid sequences, conserved pattern of amino acids and phylogenetic relationship of these toxin and nontoxin protein sequences. Sequence alignment analysis demonstrates the polarity specific molecular enrichment strategy for better system adaptivity. Occurrence of amino acid substitution is high in number in toxin sequences. In non-toxin body proteins there are less amino acid substitutions. With the help of conserved residues these proteins maintain the three-finger protein scaffold. Due to system specific adaptation toxin and non-toxin proteins exhibit a varied type of amino acid residue distribution in sequence stretch. Understanding of Natural invention scheme (recruitment of venom proteins from normal body proteins) may help us to develop futuristic engineered bio-molecules with remedial properties.

  2. Power spectral analysis of the EEG following protein malnutrition.

    PubMed

    Bronzino, J D; Stisser, P; Forbes, W B; Tracy, C; Resnick, O; Morgane, P J

    1980-01-01

    In these studies, power spectral analysis techniques were utilized to quantify the EEG obtained from rats reared on either an 8% or 25% casein diet during various vigilance states at two stages of development: (1) adulthood-90 to 120 days old; and (2) immediately after weaning-22 to 23 days old. It was found that the cortical EEG contained relatively more power in the low frequencies (ie., 0.5 to 10 Hz) for the 22-23 day old animals than for the 90-120 day old rats, especially during the slow wave sleep states-SWS1 and SWS2. Theta activity (5-8 Hz) in the hippocampus was shown to have greater power for the 22-23 day old group than for the older animals during both REM sleep and waking. Analyses of power spectral data and other indices of the frequency distribution of the hippocampal EEG indicated that those animals subjected to protein malnutrition have significantly more power in the theta band during REM sleep than the normal adult group. Since it was also noted that the hippocampal EEG obtained from the 22-23 day old group contained relatively more power in the theta band than the 90-120 day old group, the dietary treatment effect might be intrepreted as an instance of retarded development associated with protein malnutrition. Thus, a significant effect of the dietary manipulation used in the study may be largely on the system responsible for regulating theta activity.

  3. Analysis of Ribosome Inactivating Protein (RIP): A Bioinformatics Approach

    NASA Astrophysics Data System (ADS)

    Jothi, G. Edward Gnana; Majilla, G. Sahaya Jose; Subhashini, D.; Deivasigamani, B.

    2012-10-01

    In spite of the medical advances in recent years, the world is in need of different sources to encounter certain health issues.Ribosome Inactivating Proteins (RIPs) were found to be one among them. In order to get easy access about RIPs, there is a need to analyse RIPs towards constructing a database on RIPs. Also, multiple sequence alignment was done towards screening for homologues of significant RIPs from rare sources against RIPs from easily available sources in terms of similarity. Protein sequences were retrieved from SWISS-PROT and are further analysed using pair wise and multiple sequence alignment.Analysis shows that, 151 RIPs have been characterized to date. Amongst them, there are 87 type I, 37 type II, 1 type III and 25 unknown RIPs. The sequence length information of various RIPs about the availability of full or partial sequence was also found. The multiple sequence alignment of 37 type I RIP using the online server Multalin, indicates the presence of 20 conserved residues. Pairwise alignment and multiple sequence alignment of certain selected RIPs in two groups namely Group I and Group II were carried out and the consensus level was found to be 98%, 98% and 90% respectively.

  4. Deciphering primordial cyanobacterial genome functions from protein network analysis.

    PubMed

    Harel, Arye; Karkar, Slim; Cheng, Shu; Falkowski, Paul G; Bhattacharya, Debashish

    2015-03-02

    The Great Oxidation Event (GOE) ∼2.4 billion years ago resulted from the accumulation of oxygen by the ancestors of cyanobacteria [1-3]. Cyanobacteria continue to play a significant role in primary production [4] and in regulating the global marine and limnic nitrogen cycles [5, 6]. Relatively little is known, however, about the evolutionary history and gene content of primordial cyanobacteria [7, 8]. To address these issues, we used protein similarity networks [9], containing proteomes from 48 cyanobacteria as the test group, and reference proteomes from 84 microbes representing four distinct metabolic groups from most reducing to most oxidizing: methanogens, obligate anaerobes (nonmethanogenic), facultative aerobes, and obligate aerobes. These four metabolic groups represent extant bioinformatic proxies for ancient redox chemistries, extending from an anoxic origin through the GOE and ultimately to obligate aerobes [10-13]. Analysis of the network metric degree showed a strong relationship between cyanobacteria and obligate anaerobes, from which cyanobacteria presumably arose, for core functions that include translation, photosynthesis, energy conservation, and environmental interactions. These data were used to reconstruct primordial functions in cyanobacteria that included nine gene families involved in photosynthesis, hydrogenases, and proteins involved in defense from environmental stress. The presence of 60% of these genes in both reaction center I (RC-I) and RC-II-type bacteria may be explained by selective loss of either RC in the evolutionary history of some photosynthetic lineages. Finally, the network reveals that cyanobacteria occupy a unique position among prokaryotes as a hub between anaerobes and obligate aerobes.

  5. Phylogenetic analysis of the Argonaute protein family in platyhelminths.

    PubMed

    Zheng, Yadong

    2013-03-01

    Argonaute proteins (AGOs) are mediators of gene silencing via recruitment of small regulatory RNAs to induce translational regression or degradation of targeted molecules. Platyhelminths have been reported to express microRNAs but the diversity of AGOs in the phylum has not been explored. Phylogenetic relationships of members of this protein family were studied using data from six platyhelminth genomes. Phylogenetic analysis showed that all cestode and trematode AGOs, along with some triclad planarian AGOs, were grouped into the Ago subfamily and its novel sister clade, here referred to as Cluster 1. These were very distant from Piwi and Class 3 subfamilies. By contrast, a number of planarian Piwi-like AGOs formed a novel sister clade to the Piwi subfamily. Extensive sequence searching revealed the presence of an additional locus for AGO2 in the cestode Echinococcus granulosus and exon expansion in this species and E. multilocularis. The current study suggests the absence of the Piwi subfamily and Class 3 AGOs in cestodes and trematodes and the Piwi-like AGO expansion in a free-living triclad planarian and the occurrence of exon expansion prior to or during the evolution of the most-recent common ancestor of the Echinococcus species studied. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Tools for comparative protein structure modeling and analysis.

    PubMed

    Eswar, Narayanan; John, Bino; Mirkovic, Nebojsa; Fiser, Andras; Ilyin, Valentin A; Pieper, Ursula; Stuart, Ashley C; Marti-Renom, Marc A; Madhusudhan, M S; Yerkovich, Bozidar; Sali, Andrej

    2003-07-01

    The following resources for comparative protein structure modeling and analysis are described (http://salilab.org): MODELLER, a program for comparative modeling by satisfaction of spatial restraints; MODWEB, a web server for automated comparative modeling that relies on PSI-BLAST, IMPALA and MODELLER; MODLOOP, a web server for automated loop modeling that relies on MODELLER; MOULDER, a CPU intensive protocol of MODWEB for building comparative models based on distant known structures; MODBASE, a comprehensive database of annotated comparative models for all sequences detectably related to a known structure; MODVIEW, a Netscape plugin for Linux that integrates viewing of multiple sequences and structures; and SNPWEB, a web server for structure-based prediction of the functional impact of a single amino acid substitution.

  7. Fluctuation analysis of motor protein movement and single enzyme kinetics.

    PubMed Central

    Svoboda, K; Mitra, P P; Block, S M

    1994-01-01

    We studied fluctuations in the displacement of silica beads driven by single molecules of the motor protein kinesin, moving under low mechanical loads at saturating ATP concentrations. The variance in position was significantly smaller than expected for the case of stepwise movement along a regular lattice of positions with exponentially distributed intervals. The small variance suggests that two or more sequential processes with comparable reaction rates dominate the biochemical cycle. The low value is inconsistent with certain recently proposed thermal ratchet models for motor movement as well as with scenarios where the hydrolysis of a single ATP molecule leads to a cluster of several steps. Fluctuation analysis is a potential powerful tool for studying kinetic behavior whenever the output of a single enzyme can be monitored. PMID:7991536

  8. Protein profiling and phosphoprotein analysis by isoelectric focusing.

    PubMed

    Maccarrone, Giuseppina; Filiou, Michaela D

    2015-01-01

    Protein profiling enables the qualitative characterization of a proteome of interest. Phosphorylation is a post-translational modification with regulatory functions in a plethora of cell processes. We present an experimental workflow for simultaneous analysis of the proteome and phosphoproteome with no additional enrichment for phosphoproteins/phosphopeptides. Our approach is based on isoelectric focusing (IEF) which allows the separation of peptide mixtures on an immobilized pH gradient (IPG) according to their isoelectric point. Due to the negative charge of the phosphogroup, most of the phosphopeptides migrate toward acidic pH values. Peptides and phosphopeptides are then identified by mass spectrometry (MS) and phosphopeptide spectra are manually checked for the assignment of phosphorylation sites. Here, we apply this methodology to investigate synaptosome extracts from whole mouse brain. IEF-based peptide separation is an efficient method for peptide and phosphopeptide identification.

  9. Conformational analysis of proteins with a dual polarisation silicon microring.

    PubMed

    Hoste, J-W; Werquin, S; Claes, T; Bienstman, P

    2014-02-10

    Optical microresonator biosensors have proven to be a valid tool to perform affinity analysis of a biological binding event. However, when these microresonators are excited with a single optical mode they can not distinguish between a thin dense layer of biomolecules or a thick sparse layer. This means the sensor is "blind" to changes in shape of bound biomolecules. We succeeded in exciting a Silicon-on-Insulator (SOI) microring with TE and TM polarisations simultaneously by using an asymmetrical directional coupler and as such were able to separately determine the thickness and the density (or refractive index) of a bound biolayer. A proof-of-concept is given by determining both parameters of deposited dielectric layers and by analysing the conformational changes of Bovine Serum Albumin (BSA) proteins due to a change in pH of the buffer.

  10. Synaptonemal Complex Protein 3 Transcript Analysis in Breast Cancer

    PubMed Central

    MOBASHERI, Maryam Beigom; SHIRKOOHI, Reza; MODARRESSI, Mohammad Hossein

    2016-01-01

    Background: Breast cancer is the most frequent cancer in women. Cancer/Testis antigens are immunogenic proteins ectopically expressed in human neoplasms. Synaptonemal complex protein 3 (SYCP3) belongs to cancer/testis genes family involved in meiotic events and spermatogenesis. The aim of this study was to express analysis of SYCP3 in breast cancer and validate it as a breast cancer biomarker. Methods: Expression of SYCP3 transcripts in 47 breast tumors, 6 breast cancer cell lines (MCF7, SKBR3, T47D, BT474, MDA-MB-231 and MDA-MB 468), 5 normal breast and 2 testis tissues was studied by Real Time RT-PCR reaction. The reference genes phosphoglucomutase 1 and hypoxanthine guanine phosphoribosyl transferase were used as reactions normalizers. The software tool REST 2009 was applied for statistical analysis of the data. The research was conducted from Apr 2014 to August 2015 in Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Results: All of the studied breast cancer cell lines showed very high levels of SYCP3 overexpression in comparison to normal breast (P=0.001) and even to normal testis (P=0.001), except for MCF7 cell line. Breast tumors showed moderately increasing in transcript changes in comparison to normal breast. Conclusion: SYCP3 is a known testis-specific gene, but interestingly five out of six studied breast cancer of cell lines showed higher expression levels of SYCP3 in comparison to normal testis and normal breast tissues. SYCP3 has critical role in cell division with known interaction with the tumor suppressor genes, BRCA1 and BRCA2, which are critical genes in breast cancer. PMID:28053928

  11. Comparative analysis of HOG pathway proteins to generate hypotheses for functional analysis.

    PubMed

    Krantz, Marcus; Becit, Evren; Hohmann, Stefan

    2006-03-01

    Comparative genomics allows comparison of different proteins that execute presumably identical functions in different organisms. In contrast to paralogues, orthologues per definition perform the same function and interact with the same partners and, consequently, should display conservation in all these properties. We have employed 20 fungal genomes to analyse key components of the high osmolarity glycerol signalling pathway of Saccharomyces cerevisiae. Among the proteins scrutinised are a complete phosphotransfer module, a MAP kinase, two scaffold proteins, one of which is also a MAPKK, and two transcription factors. Sequence alignments, domain structure and size analysis, combined with the rich information available in the literature, allowed us to probe previous structural and functional studies and to generate hypotheses for future experimental studies. Although certain domains are too highly conserved across fungal species for meaningful comparative studies, others, like interaction domains, can be studied in closely related species. Moreover, putative functionally relevant sites for protein modifications can be identified in such comparative studies. We provide several relevant examples and present a number of previously un(der)characterised domains of potential functional significance in osmosensing and signal transduction. We propose that any functional protein analysis in fungi should make use of the unique resource that fungal genome sequences offer.

  12. A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system.

    PubMed

    Misek, David E; Kuick, Rork; Wang, Hong; Galchev, Vladimir; Deng, Bin; Zhao, Rong; Tra, John; Pisano, Michael R; Amunugama, Ravi; Allen, David; Walker, Angela K; Strahler, John R; Andrews, Philip; Omenn, Gilbert S; Hanash, Samir M

    2005-08-01

    We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.

  13. Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates

    NASA Astrophysics Data System (ADS)

    Martin, Nicholas J.; Griffiths, Rian L.; Edwards, Rebecca L.; Cooper, Helen J.

    2015-08-01

    Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2 4H] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The `contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

  14. Protein-protein interface analysis and hot spots identification for chemical ligand design.

    PubMed

    Chen, Jing; Ma, Xiaomin; Yuan, Yaxia; Pei, Jianfeng; Lai, Luhua

    2014-01-01

    Rational design for chemical compounds targeting protein-protein interactions has grown from a dream to reality after a decade of efforts. There are an increasing number of successful examples, though major challenges remain in the field. In this paper, we will first give a brief review of the available methods that can be used to analyze protein-protein interface and predict hot spots for chemical ligand design. New developments of binding sites detection, ligandability and hot spots prediction from the author's group will also be described. Pocket V.3 is an improved program for identifying hot spots in protein-protein interface using only an apo protein structure. It has been developed based on Pocket V.2 that can derive receptor-based pharmacophore model for ligand binding cavity. Given similarities and differences between the essence of pharmacophore and hot spots for guiding design of chemical compounds, not only energetic but also spatial properties of protein-protein interface are used in Pocket V.3 for dealing with protein-protein interface. In order to illustrate the capability of Pocket V.3, two datasets have been used. One is taken from ASEdb and BID having experimental alanine scanning results for testing hot spots prediction. The other is taken from the 2P2I database containing complex structures of protein-ligand binding at the original protein-protein interface for testing hot spots application in ligand design.

  15. Biochemical analysis of oligomerization of expanded polyalanine repeat proteins.

    PubMed

    Nojima, Jun; Oma, Yoko; Futai, Eugene; Sasagawa, Noboru; Kuroda, Reiko; Turk, Boris; Ishiura, Shoichi

    2009-08-01

    Many human proteins contain amino acid repeats that can form homopolymeric amino acid (HPAA) tracts. HPAA tract proteins that contain polyalanine sequences promote diseases, including oculopharyngeal muscular dystrophy. The pathological properties of these proteins develop when the repeats match or exceed approximately 20 residues. We analyzed the oligomerization of yellow fluorescent protein (YFP) and GST fusion proteins containing >20 alanine repeats by using sucrose density gradient centrifugation. YFP and GST fusion proteins having 23 polyalanine residues sedimented readily in sucrose density gradients, suggesting instability and oligomerization of proteins with an excess of 20 alanine repeats. Moreover, GST fusion proteins were resistant to trypsin digestion after oligomerization. Oligomerized artificial proteins with long polyalanine repeats may be suitable models for studying polyalanine-related diseases.

  16. Tape Stripping Technique for Stratum Corneum Protein Analysis.

    PubMed

    Clausen, Maja-Lisa; Slotved, H-C; Krogfelt, Karen A; Agner, Tove

    2016-01-28

    The aim of this study was to investigate the amount of protein in stratum corneum in atopic dermatitis (AD) patients and healthy controls, using tape stripping technique. Furthermore, to compare two different methods for protein assessment. Tape stripping was performed in AD patients and healthy controls to collect stratum corneum samples and subsequently analysed with two different methods: Squame Scan, which gives an estimate of total protein (soluble and insoluble) and Micro BCA protein determination kit which measures soluble protein. Significant differences in cumulative protein content between AD lesional, AD non-lesional and healthy control skin was found using the Squame Scan as well as the Micro BCA protein determination kit. AD patients had significantly lower amount of protein, both total protein and soluble protein compared to healthy controls. Furthermore, soluble protein formed 82% of total protein in AD lesional skin, compared to 17-24% for AD non-lesional skin and healthy control. A decreasing amount of total protein with increasing stratum corneum depth was found for all skin types. Significant differences in stratum corneum protein content between AD lesional, AD non-lesional and healthy control skin were revealed, independent of method used.

  17. Tape Stripping Technique for Stratum Corneum Protein Analysis

    PubMed Central

    Clausen, Maja-Lisa; Slotved, H.-C.; Krogfelt, Karen A.; Agner, Tove

    2016-01-01

    The aim of this study was to investigate the amount of protein in stratum corneum in atopic dermatitis (AD) patients and healthy controls, using tape stripping technique. Furthermore, to compare two different methods for protein assessment. Tape stripping was performed in AD patients and healthy controls to collect stratum corneum samples and subsequently analysed with two different methods: Squame Scan, which gives an estimate of total protein (soluble and insoluble) and Micro BCA protein determination kit which measures soluble protein. Significant differences in cumulative protein content between AD lesional, AD non-lesional and healthy control skin was found using the Squame Scan as well as the Micro BCA protein determination kit. AD patients had significantly lower amount of protein, both total protein and soluble protein compared to healthy controls. Furthermore, soluble protein formed 82% of total protein in AD lesional skin, compared to 17–24% for AD non-lesional skin and healthy control. A decreasing amount of total protein with increasing stratum corneum depth was found for all skin types. Significant differences in stratum corneum protein content between AD lesional, AD non-lesional and healthy control skin were revealed, independent of method used. PMID:26817661

  18. Mass spectrometric analysis of post-translational modifications (PTMs) and protein-protein interactions (PPIs).

    PubMed

    Ngounou Wetie, Armand G; Woods, Alisa G; Darie, Costel C

    2014-01-01

    Of the 25,000-30,000 human genes, about 2 % code for proteins. However, there are about one to two million protein entities. This is primarily due to alternative splicing and post-translational modifications (PTMs). Identifying all these modifications in one proteome at a particular time point during development or during the transition from normal to cancerous cells is a great challenge to scientists. In addition, identifying the biological significance of all these modifications, as well as their nature, such as stable versus transient modifications, is an even more challenging. Furthermore, interaction of proteins and protein isoforms that have one or more stable or transient PTMs with other proteins and protein isoforms makes the study of proteins daunting and complex. Here we review some of the strategies to study proteins, protein isoforms, protein PTMs, and protein-protein interactions (PPIs). Our goal is to provide a thorough understanding of these proteins and their isoforms, PTMs and PPIs and to shed light on the biological significance of these factors.

  19. Proteins of unknown function in the Protein Data Bank (PDB): an inventory of true uncharacterized proteins and computational tools for their analysis.

    PubMed

    Nadzirin, Nurul; Firdaus-Raih, Mohd

    2012-10-08

    Proteins of uncharacterized functions form a large part of many of the currently available biological databases and this situation exists even in the Protein Data Bank (PDB). Our analysis of recent PDB data revealed that only 42.53% of PDB entries (1084 coordinate files) that were categorized under "unknown function" are true examples of proteins of unknown function at this point in time. The remainder 1465 entries also annotated as such appear to be able to have their annotations re-assessed, based on the availability of direct functional characterization experiments for the protein itself, or for homologous sequences or structures thus enabling computational function inference.

  20. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  1. Graph spectral analysis of protein interaction network evolution.

    PubMed

    Thorne, Thomas; Stumpf, Michael P H

    2012-10-07

    We present an analysis of protein interaction network data via the comparison of models of network evolution to the observed data. We take a bayesian approach and perform posterior density estimation using an approximate bayesian computation with sequential Monte Carlo method. Our approach allows us to perform model selection over a selection of potential network growth models. The methodology we apply uses a distance defined in terms of graph spectra which captures the network data more naturally than previously used summary statistics such as the degree distribution. Furthermore, we include the effects of sampling into the analysis, to properly correct for the incompleteness of existing datasets, and have analysed the performance of our method under various degrees of sampling. We consider a number of models focusing not only on the biologically relevant class of duplication models, but also including models of scale-free network growth that have previously been claimed to describe such data. We find a preference for a duplication-divergence with linear preferential attachment model in the majority of the interaction datasets considered. We also illustrate how our method can be used to perform multi-model inference of network parameters to estimate properties of the full network from sampled data.

  2. HaloTag: a novel protein labeling technology for cell imaging and protein analysis.

    PubMed

    Los, Georgyi V; Encell, Lance P; McDougall, Mark G; Hartzell, Danette D; Karassina, Natasha; Zimprich, Chad; Wood, Monika G; Learish, Randy; Ohana, Rachel Friedman; Urh, Marjeta; Simpson, Dan; Mendez, Jacqui; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Zhu, Ji; Darzins, Aldis; Klaubert, Dieter H; Bulleit, Robert F; Wood, Keith V

    2008-06-20

    We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.

  3. Characterization of Ternary Protein Systems In Vivo with Tricolor Heterospecies Partition Analysis.

    PubMed

    Hur, Kwang-Ho; Chen, Yan; Mueller, Joachim D

    2016-03-08

    Tools and assays that characterize protein-protein interactions are of fundamental importance to biology, because protein assemblies play a critical role in the control and regulation of nearly every cellular process. The availability of fluorescent proteins has facilitated the direct and real-time observation of protein-protein interactions inside living cells, but existing methods are mostly limited to binary interactions between two proteins. Because of the scarcity of techniques capable of identifying ternary interactions, we developed tricolor heterospecies partition analysis. The technique is based on brightness analysis of fluorescence fluctuations from three fluorescent proteins that serve as protein labels. We identified three fluorescent proteins suitable for tricolor brightness experiments. In addition, we developed the theory of identifying interactions in a ternary protein system using tricolor heterospecies partition analysis. The theory was verified by experiments on well-characterized protein systems. A graphical representation of the heterospecies partition data was introduced to visualize interactions in ternary protein systems. Lastly, we performed fluorescence fluctuation experiments on cells expressing a coactivator and two nuclear receptors and applied heterospecies partition analysis to explore the interactions of this ternary protein system. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Viscosity Analysis of Dual Variable Domain Immunoglobulin Protein Solutions: Role of Size, Electroviscous Effect and Protein-Protein Interactions.

    PubMed

    Raut, Ashlesha S; Kalonia, Devendra S

    2016-01-01

    Increased solution viscosity results in difficulties in manufacturing and delivery of therapeutic protein formulations, increasing both the time and production costs, and leading to patient inconvenience. The solution viscosity is affected by the molecular properties of both the solute and the solvent. The purpose of this work was to investigate the effect of size, charge and protein-protein interactions on the viscosity of Dual Variable Domain Immunoglobulin (DVD-Ig(TM)) protein solutions. The effect of size of the protein molecule on solution viscosity was investigated by measuring intrinsic viscosity and excluded volume calculations for monoclonal antibody (mAb) and DVD-Ig(TM) protein solutions. The role of the electrostatic charge resulting in electroviscous effects for DVD-Ig(TM) protein was assessed by measuring zeta potential. Light scattering measurements were performed to detect protein-protein interactions affecting solution viscosity. DVD-Ig(TM) protein exhibited significantly higher viscosity compared to mAb. Intrinsic viscosity and excluded volume calculations indicated that the size of the molecule affects viscosity significantly at higher concentrations, while the effect was minimal at intermediate concentrations. Electroviscous contribution to the viscosity of DVD-Ig(TM) protein varied depending on the presence or absence of ions in the solution. In buffered solutions, negative k D and B 2 values indicated the presence of attractive interactions which resulted in high viscosity for DVD-Ig(TM) protein at certain pH and ionic strength conditions. Results show that more than one factor contributes to the increased viscosity of DVD-Ig(TM) protein and interplay of these factors modulates the overall viscosity behavior of the solution, especially at higher concentrations.

  5. Yeast two-hybrid analysis of jasmonate signaling proteins.

    PubMed

    Cuéllar, Amparo Pérez; Pauwels, Laurens; De Clercq, Rebecca; Goossens, Alain

    2013-01-01

    Protein-protein interaction studies are crucial to unravel how jasmonate (JA) signals are transduced. Among the different techniques available, yeast two-hybrid (Y2H) is commonly used within the JA research community to identify proteins belonging to the core JA signaling module. The technique is based on the reconstitution of a transcriptional activator that drives the reporter gene expression upon protein-protein interactions. The method is sensitive and straightforward and can be adapted for different approaches. In this chapter, we provide a detailed protocol to perform targeted Y2H assays to test known proteins and/or protein domains for direct interaction in a pairwise manner and present the possibility to study ternary protein complexes through Y3H.

  6. Generating mammalian sirtuin tools for protein-interaction analysis.

    PubMed

    Hershberger, Kathleen A; Motley, Jonathan; Hirschey, Matthew D; Anderson, Kristin A

    2013-01-01

    The sirtuins are a family of NAD(+)-dependent deacylases with important effects on aging, cancer, and metabolism. Sirtuins exert their biological effects by catalyzing deacetylation and/or deacylation reactions in which Acyl groups are removed from lysine residues of specific proteins. A current challenge is to identify specific sirtuin target proteins against the high background of acetylated proteins recently identified by proteomic surveys. New evidence indicates that bona fide sirtuin substrate proteins form stable physical associations with their sirtuin regulator. Therefore, identification of sirtuin interacting proteins could be a useful aid in focusing the search for substrates. Described here is a method for identifying sirtuin protein interactors. Employing basic techniques of molecular cloning and immunochemistry, the method describes the generation of mammalian sirtuin protein expression plasmids and their use to overexpress and immunoprecipitate sirtuins with their interacting partners. Also described is the use of the Database for Annotation, Visualization, and Integrated Discovery for interpreting the sirtuin protein-interaction data obtained.

  7. PSEA-Quant: A Protein Set Enrichment Analysis on Label-Free and Label-Based Protein Quantification Data

    PubMed Central

    2015-01-01

    The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights. PMID:25177766

  8. Sub-synaptic, multiplexed analysis of proteins reveals Fragile X related protein 2 is mislocalized in Fmr1 KO synapses

    PubMed Central

    Wang, Gordon X; Smith, Stephen J; Mourrain, Philippe

    2016-01-01

    The distribution of proteins within sub-synaptic compartments is an essential aspect of their neurological function. Current methodologies, such as electron microscopy (EM) and super-resolution imaging techniques, can provide the precise localization of proteins, but are often limited to a small number of one-time observations with narrow spatial and molecular coverage. The diversity of synaptic proteins and synapse types demands synapse analysis on a scale that is prohibitive with current methods. Here, we demonstrate SubSynMAP, a fast, multiplexed sub-synaptic protein analysis method using wide-field data from deconvolution array tomography (ATD). SubSynMAP generates probability distributions for that reveal the functional range of proteins within the averaged synapse of a particular class. This enables the differentiation of closely juxtaposed proteins. Using this method, we analyzed 15 synaptic proteins in normal and Fragile X mental retardation syndrome (FXS) model mouse cortex, and revealed disease-specific modifications of sub-synaptic protein distributions across synapse classes and cortical layers. DOI: http://dx.doi.org/10.7554/eLife.20560.001 PMID:27770568

  9. Genomic analysis of the eukaryotic protein kinase superfamily: a perspective

    PubMed Central

    Hanks, Steven K

    2003-01-01

    Protein kinases with a conserved catalytic domain make up one of the largest 'superfamilies' of eukaryotic proteins and play many key roles in biology and disease. Efforts to identify and classify all the members of the eukaryotic protein kinase superfamily have recently culminated in the mining of essentially complete human genome data. PMID:12734000

  10. Analysis of DNA-protein complexes induced by chemical carcinogens

    SciTech Connect

    Costa, M. )

    1990-11-01

    DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.

  11. Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

    PubMed

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  12. Comparative Proteome Analysis of Cryopreserved Flagella and Head Plasma Membrane Proteins from Sea Bream Spermatozoa: Effect of Antifreeze Proteins

    PubMed Central

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  13. A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition.

    PubMed

    Bourgeois, Michael; Jacquin, Françoise; Cassecuelle, Florence; Savois, Vincent; Belghazi, Maya; Aubert, Grégoire; Quillien, Laurence; Huart, Myriam; Marget, Pascal; Burstin, Judith

    2011-05-01

    Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

    PubMed

    Merabet, Samir; Litim-Mecheri, Isma; Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-10-01

    Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

  15. Mutational analysis of the major coat protein of M13 identifies residues that control protein display.

    PubMed Central

    Weiss, G. A.; Wells, J. A.; Sidhu, S. S.

    2000-01-01

    We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487-495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities. PMID:10794407

  16. Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

    PubMed Central

    Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K.; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-01-01

    Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. PMID:22046139

  17. Construction and Structural Analysis of Tethered Lipid Bilayer Containing Photosynthetic Antenna Proteins for Functional Analysis

    SciTech Connect

    Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu; Sugiura, Ryuta; Sasaki, Nobuaki; Misawa, Nobuo; Tero, Ryugo; Urisu, Tsuneo; Gardiner, Alastair T.; Cogdell, Richard J.; Hashimoto, Hideki; Nango, Mamoru

    2011-07-11

    The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAY), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

  18. Statistical database analysis of the role of loop dynamics for protein-protein complex formation and allostery.

    PubMed

    Gu, Yina; Li, Da-Wei; Brüschweiler, Rafael

    2017-06-15

    Protein loops show rich conformational dynamics properties on a wide range of timescales as they play an essential role for many cellular functions during protein-protein interactions and recognition processes. However, little is known about the detail behavior of loops upon protein binding including allostery. We report the loop motions and their dominant timescales for a library of 230 proteins that form protein-protein complexes using the ToeLoop predictor of loop dynamics. We applied the analysis to proteins in both their complex and free state and relate specific loop properties to their role in protein recognition. We observe a strong tendency of loops that move on relatively slow timescales of tens of ns to sub-μs to be directly involved in binding and recognition processes. Complex formation leads to a significant reduction in loop flexibility at the binding interface, but in a number of cases it can also trigger increased flexibility in distal loops in response to allosteric conformational changes. The importance of loop dynamics and allostery is highlighted by a case study of an antibody-antigen complex. Furthermore, we explored the relationship between loop dynamics and experimental binding affinities and found that a prevalence of high loop rigidity at the binding interface is an indicator of increased binding strength. http://spin.ccic.ohio-state.edu/index.php/toeloopppi. bruschweiler.1@osu.edu. Supplementary data are available at Bioinformatics online.

  19. Analysis of crystallization data in the Protein Data Bank.

    PubMed

    Kirkwood, Jobie; Hargreaves, David; O'Keefe, Simon; Wilson, Julie

    2015-10-01

    The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored.

  20. Analysis of crystallization data in the Protein Data Bank

    SciTech Connect

    Kirkwood, Jobie; Hargreaves, David; O’Keefe, Simon; Wilson, Julie

    2015-09-23

    In a large-scale study using data from the Protein Data Bank, some of the many reported findings regarding the crystallization of proteins were investigated. The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored.

  1. Inference of a Geminivirus-Host Protein-Protein Interaction Network through Affinity Purification and Mass Spectrometry Analysis.

    PubMed

    Wang, Liping; Ding, Xue; Xiao, Jiajing; Jiménez-Gόngora, Tamara; Liu, Renyi; Lozano-Durán, Rosa

    2017-09-25

    Viruses reshape the intracellular environment of their hosts, largely through protein-protein interactions, to co-opt processes necessary for viral infection and interference with antiviral defences. Due to genome size constraints and the concomitant limited coding capacity of viruses, viral proteins are generally multifunctional and have evolved to target diverse host proteins. Inference of the virus-host interaction network can be instrumental for understanding how viruses manipulate the host machinery and how re-wiring of specific pathways can contribute to disease. Here, we use affinity purification and mass spectrometry analysis (AP-MS) to define the global landscape of interactions between the geminivirus Tomato yellow leaf curl virus (TYLCV) and its host Nicotiana benthamiana. For this purpose, we expressed tagged versions of each of TYLCV-encoded proteins (C1/Rep, C2/TrAP, C3/REn, C4, V2, and CP) in planta in the presence of the virus. Using a quantitative scoring system, 728 high-confidence plant interactors were identified, and the interaction network of each viral protein was inferred; TYLCV-targeted proteins are more connected than average, and connect with other proteins through shorter paths, which would allow the virus to exert large effects with few interactions. Comparative analyses of divergence patterns between N. benthamiana and potato, a non-host Solanaceae, showed evolutionary constraints on TYLCV-targeted proteins. Our results provide a comprehensive overview of plant proteins targeted by TYLCV during the viral infection, which may contribute to uncovering the underlying molecular mechanisms of plant viral diseases and provide novel potential targets for anti-viral strategies and crop engineering. Interestingly, some of the TYLCV-interacting proteins appear to be convergently targeted by other pathogen effectors, which suggests a central role for these proteins in plant-pathogen interactions, and pinpoints them as potential targets to

  2. Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals.

    PubMed

    Arakaki, Atsushi; Kikuchi, Daiki; Tanaka, Masayoshi; Yamagishi, Ayana; Yoda, Takuto; Matsunaga, Tadashi

    2016-10-15

    The magnetosome is an organelle specialized for inorganic magnetite crystal synthesis in magnetotactic bacteria. The complex mechanism of magnetosome formation is regulated by magnetosome proteins in a stepwise manner. Protein localization is a key step for magnetosome development; however, a global study of magnetosome protein localization remains to be conducted. Here, we comparatively analyzed the subcellular localization of a series of green fluorescent protein (GFP)-tagged magnetosome proteins. The protein localizations were categorized into 5 groups (short-length linear, middle-length linear, long-length linear, cell membrane, and intracellular dispersing), which were related to the protein functions. Mms6, which regulates magnetite crystal growth, localized along magnetosome chain structures under magnetite-forming (microaerobic) conditions but was dispersed in the cell under nonforming (aerobic) conditions. Correlative fluorescence and electron microscopy analyses revealed that Mms6 preferentially localized to magnetosomes enclosing magnetite crystals. We suggest that a highly organized spatial regulation mechanism controls magnetosome protein localization during magnetosome formation in magnetotactic bacteria. Magnetotactic bacteria synthesize magnetite (Fe3O4) nanocrystals in a prokaryotic organelle called the magnetosome. This organelle is formed using various magnetosome proteins in multiple steps, including vesicle formation, magnetosome alignment, and magnetite crystal formation, to provide compartmentalized nanospaces for the regulation of iron concentrations and redox conditions, enabling the synthesis of a morphologically controlled magnetite crystal. Thus, to rationalize the complex organelle development, the localization of magnetosome proteins is considered to be highly regulated; however, the mechanisms remain largely unknown. Here, we performed comparative localization analysis of magnetosome proteins that revealed the presence of a spatial

  3. Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals

    PubMed Central

    Arakaki, Atsushi; Kikuchi, Daiki; Tanaka, Masayoshi; Yamagishi, Ayana; Yoda, Takuto

    2016-01-01

    ABSTRACT The magnetosome is an organelle specialized for inorganic magnetite crystal synthesis in magnetotactic bacteria. The complex mechanism of magnetosome formation is regulated by magnetosome proteins in a stepwise manner. Protein localization is a key step for magnetosome development; however, a global study of magnetosome protein localization remains to be conducted. Here, we comparatively analyzed the subcellular localization of a series of green fluorescent protein (GFP)-tagged magnetosome proteins. The protein localizations were categorized into 5 groups (short-length linear, middle-length linear, long-length linear, cell membrane, and intracellular dispersing), which were related to the protein functions. Mms6, which regulates magnetite crystal growth, localized along magnetosome chain structures under magnetite-forming (microaerobic) conditions but was dispersed in the cell under nonforming (aerobic) conditions. Correlative fluorescence and electron microscopy analyses revealed that Mms6 preferentially localized to magnetosomes enclosing magnetite crystals. We suggest that a highly organized spatial regulation mechanism controls magnetosome protein localization during magnetosome formation in magnetotactic bacteria. IMPORTANCE Magnetotactic bacteria synthesize magnetite (Fe3O4) nanocrystals in a prokaryotic organelle called the magnetosome. This organelle is formed using various magnetosome proteins in multiple steps, including vesicle formation, magnetosome alignment, and magnetite crystal formation, to provide compartmentalized nanospaces for the regulation of iron concentrations and redox conditions, enabling the synthesis of a morphologically controlled magnetite crystal. Thus, to rationalize the complex organelle development, the localization of magnetosome proteins is considered to be highly regulated; however, the mechanisms remain largely unknown. Here, we performed comparative localization analysis of magnetosome proteins that revealed the

  4. The essential and downstream common proteins of amyotrophic lateral sclerosis: A protein-protein interaction network analysis

    PubMed Central

    Chen, Le; Heckman, C. J.

    2017-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a devastative neurodegenerative disease characterized by selective loss of motoneurons. While several breakthroughs have been made in identifying ALS genetic defects, the detailed molecular mechanisms are still unclear. These genetic defects involve in numerous biological processes, which converge to a common destiny: motoneuron degeneration. In addition, the common comorbid Frontotemporal Dementia (FTD) further complicates the investigation of ALS etiology. In this study, we aimed to explore the protein-protein interaction network built on known ALS-causative genes to identify essential proteins and common downstream proteins between classical ALS and ALS+FTD (classical ALS + ALS/FTD) groups. The results suggest that classical ALS and ALS+FTD share similar essential protein set (VCP, FUS, TDP-43 and hnRNPA1) but have distinctive functional enrichment profiles. Thus, disruptions to these essential proteins might cause motoneuron susceptible to cellular stresses and eventually vulnerable to proteinopathies. Moreover, we identified a common downstream protein, ubiquitin-C, extensively interconnected with ALS-causative proteins (22 out of 24) which was not linked to ALS previously. Our in silico approach provides the computational background for identifying ALS therapeutic targets, and points out the potential downstream common ground of ALS-causative mutations. PMID:28282387

  5. The essential and downstream common proteins of amyotrophic lateral sclerosis: A protein-protein interaction network analysis.

    PubMed

    Mao, Yimin; Kuo, Su-Wei; Chen, Le; Heckman, C J; Jiang, M C

    2017-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a devastative neurodegenerative disease characterized by selective loss of motoneurons. While several breakthroughs have been made in identifying ALS genetic defects, the detailed molecular mechanisms are still unclear. These genetic defects involve in numerous biological processes, which converge to a common destiny: motoneuron degeneration. In addition, the common comorbid Frontotemporal Dementia (FTD) further complicates the investigation of ALS etiology. In this study, we aimed to explore the protein-protein interaction network built on known ALS-causative genes to identify essential proteins and common downstream proteins between classical ALS and ALS+FTD (classical ALS + ALS/FTD) groups. The results suggest that classical ALS and ALS+FTD share similar essential protein set (VCP, FUS, TDP-43 and hnRNPA1) but have distinctive functional enrichment profiles. Thus, disruptions to these essential proteins might cause motoneuron susceptible to cellular stresses and eventually vulnerable to proteinopathies. Moreover, we identified a common downstream protein, ubiquitin-C, extensively interconnected with ALS-causative proteins (22 out of 24) which was not linked to ALS previously. Our in silico approach provides the computational background for identifying ALS therapeutic targets, and points out the potential downstream common ground of ALS-causative mutations.

  6. Immunochemical analysis of acetaminophen covalent binding to proteins. Partial characterization of the major acetaminophen-binding liver proteins.

    PubMed

    Bartolone, J B; Birge, R B; Sparks, K; Cohen, S D; Khairallah, E A

    1988-12-15

    A sensitive immunoassay for detecting acetaminophen (APAP) bound to proteins was developed using an affinity purified antibody directed against the N-acetylated end of the APAP molecule. Western blots of electrophoretically resolved liver proteins taken from mice given an hepatotoxic dose of APAP demonstrated that nearly 85% of the total detectable protein-bound APAP was covalently associated with proteins of 44 and 58 kD. Pretreatment of liver extracts with the sulfhydryl-specific reagent, N-ethylmaleimide (NEM), prior to derivatization with the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), greatly reduced immunochemically detectable APAP-protein adducts and indicated that the antibody detects protein-thiol conjugates of APAP. To investigate the basis of the binding selectivity in vivo, a variety of systems which yielded APAP-protein adducts were analyzed. Systems which activate APAP enzymatically, as in hepatocyte suspensions or in post-mitochondrial (S9) fractions fortified with an NADPH-regenerating system, resulted in a protein binding profile similar to that produced in vivo. Conversely, when extracts or cells were treated with chemically synthesized NAPQI, an alternative protein binding profile was obtained. Two-dimensional electrophoretic analysis of the reduced protein thiol (PSH) content of liver proteins using [3H]NEM labeling revealed that the 58 kD APAP-binding proteins were rich in PSH, whereas the major 44 kD binding protein had virtually no detectable PSH. Many PSH-rich proteins that were not arylated in vivo did bind NAPQI in vitro. However, the 44 kD proteins were not arylated when chemically synthesized NAPQI was added to homogenates or cell suspensions. The present data further suggest that, in addition to the amount and reactivity of free protein sulfhydryls, the cellular localization with respect to the cytochrome P-450 activation site may influence the susceptibility of proteins to NAPQI binding. These findings signal

  7. Are Proteins Just Coiled Cords? Local and Global Analysis of Contact Maps Reveals the Backbone-Dependent Nature of Proteins.

    PubMed

    Santoni, Daniele; Paci, Paola; Di Paola, Luisa; Giuliani, Alessandro

    2016-01-01

    In this work, we present an extensive analysis of protein contact network topology applied to a wide data set. We extended the concept of degree distribution to graphlets, describing local connectivity patterns. We compared results to those derived from artificial networks of the same size (number of nodes), reproducing the average degree of each protein network. The artificial networks resemble the coiling of immaterial cords and we tried to understand if they could catch the protein structure topology upon the sole constraint of backbone (cord). We found a surprisingly similar pattern for local topological descriptors (graphlets distribution) while real proteins and cords differ at large extent in the global topological invariant average shortest path that presumably catches the systemic nature of protein and the non negligible encumbrance of backbone (residues steric hindrance). We demonstrated average shortest path to link polymer length and physical size of the molecule, and its minimization plays the role of `target function` of folding process.

  8. The emerging process of Top Down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput†

    PubMed Central

    Kellie, John F.; Tran, John C.; Lee, Ji Eun; Ahlf, Dorothy R.; Thomas, Haylee M.; Ntai, Ioanna; Catherman, Adam D.; Durbin, Kenneth R.; Zamdborg, Leonid; Vellaichamy, Adaikkalam; Thomas, Paul M.

    2011-01-01

    Top Down mass spectrometry (MS) has emerged as an alternative to common Bottom Up strategies for protein analysis. In the Top Down approach, intact proteins are fragmented directly in the mass spectrometer to achieve both protein identification and characterization, even capturing information on combinatorial post-translational modifications. Just in the past two years, Top Down MS has seen incremental advances in instrumentation and dedicated software, and has also experienced a major boost from refined separations of whole proteins in complex mixtures that have both high recovery and reproducibility. Combined with steadily advancing commercial MS instrumentation and data processing, a high-throughput workflow covering intact proteins and polypeptides up to 70 kDa is directly visible in the near future. PMID:20711533

  9. Proteome Analysis. Novel Proteins Identified at the Peribacteroid Membrane from Lotus japonicus Root Nodules1

    PubMed Central

    Wienkoop, Stefanie; Saalbach, Gerhard

    2003-01-01

    The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia. The model legume Lotus japonicus was chosen to study the proteins present at the PBM by proteome analysis. PBM was purified from root nodules by an aqueous polymer two-phase system. Extracted proteins were subjected to a global trypsin digest. The peptides were separated by nanoscale liquid chromatography and analyzed by tandem mass spectrometry. Searching the nonredundant protein database and the green plant expressed sequence tag database using the tandem mass spectrometry data identified approximately 94 proteins, a number far exceeding the number of proteins reported for the PBM hitherto. In particular, a number of membrane proteins like transporters for sugars and sulfate; endomembrane-associated proteins such as GTP-binding proteins and vesicle receptors; and proteins involved in signaling, for example, receptor kinases, calmodulin, 14-3-3 proteins, and pathogen response-related proteins, including a so-called HIR protein, were detected. Several ATPases and aquaporins were present, indicating a more complex situation than previously thought. In addition, the unexpected presence of a number of proteins known to be located in other compartments was observed. Two characteristic protein complexes obtained from native gel electrophoresis of total PBM proteins were also analyzed. Together, the results identified specific proteins at the PBM involved in important physiological processes and localized proteins known from nodule-specific expressed sequence tag databases to the PBM. PMID:12644660

  10. Graph theoretic network analysis reveals protein pathways underlying cell death following neurotropic viral infection

    PubMed Central

    Ghosh, Sourish; Kumar, G. Vinodh; Basu, Anirban; Banerjee, Arpan

    2015-01-01

    Complex protein networks underlie any cellular function. Certain proteins play a pivotal role in many network configurations, disruption of whose expression proves fatal to the cell. An efficient method to tease out such key proteins in a network is still unavailable. Here, we used graph-theoretic measures on protein-protein interaction data (interactome) to extract biophysically relevant information about individual protein regulation and network properties such as formation of function specific modules (sub-networks) of proteins. We took 5 major proteins that are involved in neuronal apoptosis post Chandipura Virus (CHPV) infection as seed proteins in a database to create a meta-network of immediately interacting proteins (1st order network). Graph theoretic measures were employed to rank the proteins in terms of their connectivity and the degree upto which they can be organized into smaller modules (hubs). We repeated the analysis on 2nd order interactome that includes proteins connected directly with proteins of 1st order. FADD and Casp-3 were connected maximally to other proteins in both analyses, thus indicating their importance in neuronal apoptosis. Thus, our analysis provides a blueprint for the detection and validation of protein networks disrupted by viral infections. PMID:26404759

  11. Sequence Analysis and Evolutionary Studies of Reelin Proteins

    PubMed Central

    Manoharan, Malini; Muhammad, Sayyed Auwn; Sowdhamini, Ramanathan

    2015-01-01

    The reelin gene is conserved across many vertebrate species, including humans. The protein product of this gene plays several important roles in early brain development and regulation of neural network plasticity of a matured brain structure. With an extended structure of 3461 amino acid sequences, consisting of eight reelin repeats, the human reelin sequence stands out as an exceptional model for evolutionary studies. In this study, sequence analysis of the human reelin and its homologues and reelin sequences from 104 other species is described in detail. Interesting sequence conservation patterns of individual repeats have been highlighted. Sequence phylogeny of the reelin sequences indicates a pattern similar to the evolution of the species, thereby serving as a highly conserved family for evolutionary purposes. Multiple sequence alignment of different reelin domain repeats, derived from homologues, suggests specific functions for individual repeats and high sequence conservation across reelin repeats from different organisms, albeit with few unusual domain architectures. A three-dimensional structural model of the full-length human reelin is now available that provides clues on residues at the dimer interface. PMID:26715843

  12. A general sequence processing and analysis program for protein engineering.

    PubMed

    Stafford, Ryan L; Zimmerman, Erik S; Hallam, Trevor J; Sato, Aaron K

    2014-10-27

    Protein engineering projects often amass numerous raw DNA sequences, but no readily available software combines sequence processing and activity correlation required for efficient lead identification. XLibraryDisplay is an open source program integrated into Microsoft Excel for Windows that automates batch sequence processing via a simple step-by-step, menu-driven graphical user interface. XLibraryDisplay accepts any DNA template which is used as a basis for trimming, filtering, translating, and aligning hundreds to thousands of sequences (raw, FASTA, or Phred PHD file formats). Key steps for library characterization through lead discovery are available including library composition analysis, filtering by experimental data, graphing and correlating to experimental data, alignment to structural data extracted from PDB files, and generation of PyMOL visualization scripts. Though larger data sets can be handled, the program is best suited for analyzing approximately 10 000 or fewer leads or naïve clones which have been characterized using Sanger sequencing and other experimental approaches. XLibraryDisplay can be downloaded for free from sourceforge.net/projects/xlibrarydisplay/ .

  13. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  14. Structural Analysis of Protein-Protein Interactions in Type I Polyketide Synthases

    PubMed Central

    Xu, Wei; Qiao, Kangjian; Tang, Yi

    2013-01-01

    Polyketide synthases (PKSs) are responsible for synthesizing a myriad of natural products with agricultural, medicinal relevance. The PKSs consist of multiple functional domains of which each can catalyze a specified chemical reaction leading to the synthesis of polyketides. Biochemical studies showed that protein-substrate and protein-protein interactions play crucial roles in these complex regio-/stereo- selective biochemical processes. Recent developments on X-ray crystallography and protein NMR techniques have allowed us to understand the biosynthetic mechanism of these enzymes from their structures. These structural studies have facilitated the elucidation of sequence-function relationship of PKSs and will ultimately contribute to the prediction of product structure. This review will focus on the current knowledge of type I PKS structures and the protein-protein interactions in this system. PMID:23249187

  15. Introducing Students to Protein Analysis Techniques: Separation and Comparative Analysis of Gluten Proteins in Various Wheat Strains

    ERIC Educational Resources Information Center

    Pirinelli, Alyssa L.; Trinidad, Jonathan C.; Pohl, Nicola L. B.

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) is commonly taught in undergraduate laboratory classes as a traditional method to analyze proteins. An experiment has been developed to teach these basic protein gel skills in the context of gluten protein isolation from various types of wheat flour. A further goal is to relate this technique to current…

  16. Introducing Students to Protein Analysis Techniques: Separation and Comparative Analysis of Gluten Proteins in Various Wheat Strains

    ERIC Educational Resources Information Center

    Pirinelli, Alyssa L.; Trinidad, Jonathan C.; Pohl, Nicola L. B.

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) is commonly taught in undergraduate laboratory classes as a traditional method to analyze proteins. An experiment has been developed to teach these basic protein gel skills in the context of gluten protein isolation from various types of wheat flour. A further goal is to relate this technique to current…

  17. Stable Isotope Labeling Strategy for Protein-Ligand Binding Analysis in Multi-Component Protein Mixtures

    NASA Astrophysics Data System (ADS)

    DeArmond, Patrick D.; West, Graham M.; Huang, Hai-Tsang; Fitzgerald, Michael C.

    2011-03-01

    Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H{2/16}O2 and H{2/18}O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

  18. Computational protein structure modeling and analysis of UV-B stress protein in Synechocystis PCC 6803.

    PubMed

    Rahman, Md Akhlaqur; Chaturvedi, Navaneet; Sinha, Sukrat; Pandey, Paras Nath; Gupta, Dwijendra Kumar; Sundaram, Shanthy; Tripathi, Ashutosh

    2013-01-01

    This study focuses on Ultra Violet stress (UVS) gene product which is a UV stress induced protein from cyanobacteria, Synechocystis PCC 6803. Three dimensional structural modeling of target UVS protein was carried out by homology modeling method. 3F2I pdb from Nostoc sp. PCC 7120 was selected as a suitable template protein structure. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in modeled UV-B stress protein. The top five probable ligand binding sites were predicted and the common binding residues between target and template protein was analyzed. It has been validated for the first time that modeled UVS protein structure from Synechocystis PCC 6803 was structurally and functionally similar to well characterized UVS protein of another cyanobacterial species, Nostoc sp PCC 7120 because of having same structural motif and fold with similar protein topology and function. Investigations revealed that UVS protein from Synechocystis sp. might play significant role during ultraviolet resistance. Thus, it could be a potential biological source for remediation for UV induced stress.

  19. Extraction of intracellular protein from Glaciozyma antarctica for proteomics analysis

    NASA Astrophysics Data System (ADS)

    Faizura, S. Nor; Farahayu, K.; Faizal, A. B. Mohd; Asmahani, A. A. S.; Amir, R.; Nazalan, N.; Diba, A. B. Farah; Muhammad, M. Nor; Munir, A. M. Abdul

    2013-11-01

    Two preparation methods of crude extracts of psychrophilic yeast Glaciozyma antarctica were compared in order to obtain a good recovery of intracellular proteins. Extraction with mechanical procedures using sonication was found to be more effective for obtaining good yield compare to alkaline treatment method. The procedure is simple, rapid, and produce better yield. A total of 52 proteins were identified by combining both extraction methods. Most of the proteins identified in this study involves in the metabolic process including glycolysis pathway, pentose phosphate pathway, pyruyate decarboxylation and also urea cyle. Several chaperons were identified including probable cpr1-cyclophilin (peptidylprolyl isomerase), macrolide-binding protein fkbp12 and heat shock proteins which were postulate to accelerate proper protein folding. Characteristic of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

  20. Analysis of Structured and Intrinsically Disordered Regions of Transmembrane Proteins

    PubMed Central

    Xue, Bin; Li, Liwei; Meroueh, Samy O.; Uversky, Vladimir N.; Dunker, A. Keith

    2010-01-01

    Integral membrane proteins display two major types of transmembrane structures, helical bundles and beta barrels. The main functional roles of transmembrane proteins are the transport of small molecules and cell signaling, and sometimes these two roles are coupled. For cytosolic, water-soluble proteins, signaling and regulatory functions are often carried out by intrinsically disordered regions. Our long range goal is to determine whether integral membrane proteins likewise often use disordered regions for signaling and regulation. Here we carried out a systematic bioinformatics investigation of intrinsically disordered regions obtained from integral membrane proteins for which crystal structures have been determined, and for which the intrinsic disorder was identified as missing electron density. We found 120 disorder-containing integral membrane proteins having a total of 33,675 residues, with 3209 of the residues distributed among 240 different disordered regions. These disordered regions were compared with those obtained from water-soluble proteins with regard to their amino acid compositional biases, and with regard to accuracies of various disorder predictors. The results of these analyses show that the disordered regions from helical bundle integral membrane proteins, those from beta barrel integral membrane proteins, and those from water soluble proteins all exhibit statistically distinct amino acid compositional biases. Despite these differences in composition, current algorithms make reasonably accurate predictions of disorder for these membrane proteins. Although the small size of the current data sets are limiting, these results suggest that developing new predictors that make use of data from disordered regions in helical bundles and beta barrels, especially as these datasets increase in size, will likely lead to significantly more accurate disorder predictions for these two classes of integral membrane proteins. PMID:19585006

  1. Protein crowding on biomembranes: Analysis of contour instabilities

    NASA Astrophysics Data System (ADS)

    Manyuhina, O. V.

    2014-08-01

    Collective behavior of proteins on biomembranes is usually studied within the spontaneous curvature model. Here we consider an alternative phenomenological approach, which accounts consistently for partial ordering of proteins as well as the anchoring forces exerted on a membrane by layer of proteins. We show analytically that such anisotropic interactions can drive membrane bending, resulting in nontrivial equilibrium morphologies. The predicted instabilities can advance our conceptual understanding of physical mechanisms behind collective phenomena in biological systems, in particular those with inherent anisotropy.

  2. Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors.

    PubMed

    Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

    2012-06-01

    WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.

  3. Ions and the protein surface revisited: extensive molecular dynamics simulations and analysis of protein structures in alkali-chloride solutions.

    PubMed

    Friedman, Ran

    2011-07-28

    Proteins interact with ions in various ways. The surface of proteins has an innate capability to bind ions, and it is also influenced by the screening of the electrostatic potential owing to the presence of salts in the bulk solution. Alkali metal ions and chlorides interact with the protein surface, but such interactions are relatively weak and often transient. In this paper, computer simulations and analysis of protein structures are used to characterize the interactions between ions and the protein surface. The results show that the ion-binding properties of protein residues are highly variable. For example, alkali metal ions are more often associated with aspartate residues than with glutamates, whereas chlorides are most likely to be located near arginines. When comparing NaCl and KCl solutions, it was found that certain surface residues attract the anion more strongly in NaCl. This study demonstrates that protein-salt interactions should be accounted for in the planning and execution of experiments and simulations involving proteins, particularly if subtle structural details are sought after. © 2011 American Chemical Society

  4. Development of a pipeline for automated, high-throughput analysis of paraspeckle proteins reveals specific roles for importin α proteins

    PubMed Central

    Major, Andrew T.; Miyamoto, Yoichi; Lo, Camden Y.; Jans, David A.; Loveland, Kate L.

    2017-01-01

    We developed a large-scale, unbiased analysis method to measure how functional variations in importin (IMP) α2, IMPα4 and IMPα6 each influence PSPC1 and SFPQ nuclear accumulation and their localization to paraspeckles. This addresses the hypothesis that individual IMP protein activities determine cargo nuclear access to influence cell fate outcomes. We previously demonstrated that modulating IMPα2 levels alters paraspeckle protein 1 (PSPC1) nuclear accumulation and affects its localization into a subnuclear domain that affects RNA metabolism and cell survival, the paraspeckle. An automated, high throughput, image analysis pipeline with customisable outputs was created using Imaris software coupled with Python and R scripts; this allowed non-subjective identification of nuclear foci, nuclei and cells. HeLa cells transfected to express exogenous full-length and transport-deficient IMPs were examined using SFPQ and PSPC1 as paraspeckle markers. Thousands of cells and >100,000 nuclear foci were analysed in samples with modulated IMPα functionality. This analysis scale enabled discrimination of significant differences between samples where paraspeckles inherently display broad biological variability. The relative abundance of paraspeckle cargo protein(s) and individual IMPs each influenced nuclear foci numbers and size. This method provides a generalizable high throughput analysis platform for investigating how regulated nuclear protein transport controls cellular activities. PMID:28240251

  5. Development of a pipeline for automated, high-throughput analysis of paraspeckle proteins reveals specific roles for importin α proteins.

    PubMed

    Major, Andrew T; Miyamoto, Yoichi; Lo, Camden Y; Jans, David A; Loveland, Kate L

    2017-02-27

    We developed a large-scale, unbiased analysis method to measure how functional variations in importin (IMP) α2, IMPα4 and IMPα6 each influence PSPC1 and SFPQ nuclear accumulation and their localization to paraspeckles. This addresses the hypothesis that individual IMP protein activities determine cargo nuclear access to influence cell fate outcomes. We previously demonstrated that modulating IMPα2 levels alters paraspeckle protein 1 (PSPC1) nuclear accumulation and affects its localization into a subnuclear domain that affects RNA metabolism and cell survival, the paraspeckle. An automated, high throughput, image analysis pipeline with customisable outputs was created using Imaris software coupled with Python and R scripts; this allowed non-subjective identification of nuclear foci, nuclei and cells. HeLa cells transfected to express exogenous full-length and transport-deficient IMPs were examined using SFPQ and PSPC1 as paraspeckle markers. Thousands of cells and >100,000 nuclear foci were analysed in samples with modulated IMPα functionality. This analysis scale enabled discrimination of significant differences between samples where paraspeckles inherently display broad biological variability. The relative abundance of paraspeckle cargo protein(s) and individual IMPs each influenced nuclear foci numbers and size. This method provides a generalizable high throughput analysis platform for investigating how regulated nuclear protein transport controls cellular activities.

  6. Photoacoustic analysis of proteins: volumetric signals and fluorescence quantum yields.

    PubMed Central

    Kurian, E; Prendergast, F G; Small, J R

    1997-01-01

    A series of proteins has been examined using time-resolved, pulsed-laser volumetric photoacoustic spectroscopy. Photoacoustic waveforms were collected to measure heat release for calculation of fluorescence quantum yields, and to explore the possibility of photoinduced nonthermal volume changes occurring in these protein samples. The proteins studied were the green fluorescent protein (GFP); intestinal fatty acid binding protein (IFABP), and adipocyte lipid-binding protein (ALBP), each labeled noncovalently with 1-anilinonaphthalene-8-sulfonate (1,8-ANS) and covalently with 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan); and acrylodan-labeled IFABP and ALBP with added oleic acid. Of this group of proteins, only the ALBP labeled with 1,8-ANS showed significant nonthermal volume changes at the beta = 0 temperature (approximately 3.8 degrees C) for the buffer used (10 mM Tris-HCI, pH 7.5) (beta is the thermal cubic volumetric expansion coefficient). For all of the proteins except for acrylodan-labeled IFABP, the fluorescence quantum yields calculated assuming simple energy conservation were anomalously high, i.e., the apparent heat signals were lower than those predicted from independent fluorescence measurements. The consistent anomalies suggest that the low photoacoustic signals may be characteristic of fluorophores buried in proteins, and that photoacoustic signals derive in part from the microenvironment of the absorbing chromophore. Images FIGURE 1 PMID:9199809

  7. To What Extent is FAIMS Beneficial in the Analysis of Proteins?

    PubMed

    Cooper, Helen J

    2016-04-01

    High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, is emerging as a tool for biomolecular analysis. In this article, the benefits and limitations of FAIMS for protein analysis are discussed. The principles and mechanisms of FAIMS separation of ions are described, and the differences between FAIMS and conventional ion mobility spectrometry are detailed. Protein analysis is considered from both the top-down (intact proteins) and the bottom-up (proteolytic peptides) perspective. The roles of FAIMS in the analysis of complex mixtures of multiple intact proteins and in the analysis of multiple conformers of a single protein are assessed. Similarly, the application of FAIMS in proteomics and targeted analysis of peptides are considered.

  8. In Vitro Dynamic Visualization Analysis of Fluorescently Labeled Minor Capsid Protein IX and Core Protein V by Simultaneous Detection

    PubMed Central

    Ugai, Hideyo; Wang, Minghui; Le, Long P.; Matthews, David A.; Yamamoto, Masato; Curiel, David T.

    2009-01-01

    Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation to human clinical trials requires careful evaluation of these viral characteristics. While the function of the adenovirus proteins have been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints which prevent adequate tracking of the adenovirus particles after infection. Fluorescent labeling of the adenoviral particles is one new strategy designed to directly analyze dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling technique of adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in live analysis of adenovirus as compared to a single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX-mRFP1) and a green fluorescent minor core protein V (pV-EGFP), resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, the growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed approximately 150-fold reduced production of the viral progeny at 48 hours post-infection (h.p.i.) as compared to Ad5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and the nucleolus, respectively, at 18 h.p.i. These proteins were observed in the nucleus during the late stage of infection and the relocalization of the proteins was observed in an adenoviral replication-dependent manner. These results indicate that the simultaneous detection of adenovirus using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells, and monitoring viral spread, which will be required for complete evaluation of oncolytic adenoviruses. PMID:19853616

  9. Analysis of nitroxide spin label motion in a protein protein complex using multiple frequency EPR spectroscopy

    NASA Astrophysics Data System (ADS)

    White, G. F.; Ottignon, L.; Georgiou, T.; Kleanthous, C.; Moore, G. R.; Thomson, A. J.; Oganesyan, V. S.

    2007-04-01

    X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.

  10. Analysis of xylem sap proteins from Brassica napus

    PubMed Central

    Kehr, Julia; Buhtz, Anja; Giavalisco, Patrick

    2005-01-01

    Background Substance transport in higher land plants is mediated by vascular bundles, consisting of phloem and xylem strands that interconnect all plant organs. While the phloem mainly allocates photoassimilates, the role of the xylem is the transport of water and inorganic nutrients from roots to all aerial plant parts. Only recently it was noticed that in addition to mineral salts, xylem sap contains organic nutrients and even proteins. Although these proteins might have important impact on the performance of above-ground organs, only a few of them have been identified so far and their physiological functions are still unclear. Results We used root-pressure xylem exudate, collected from cut Brassica napus stems, to extract total proteins. These protein preparations were then separated by high-resolution two-dimensional gel electrophoresis (2-DE). After individual tryptic digests of the most abundant coomassie-stained protein spots, partial peptide sequence information was deduced from tandem mass spectrometric (MS/MS) fragmentation spectra and subsequently used for protein identifications by database searches. This approach resulted in the identification of 69 proteins. These identifications include different proteins potentially involved in defence-related reactions and cell wall metabolism. Conclusion This study provides a comprehensive overview of the most abundant proteins present in xylem sap of Brassica napus. A number of 69 proteins could be identified from which many previously were not known to be localized to this compartment in any other plant species. Since Brassica napus, a close relative of the fully sequenced model plant Arabidopsis thaliana, was used as the experimental system, our results provide a large number of candidate proteins for directed molecular and biochemical analyses of the physiological functions of the xylem under different environmental and developmental conditions. This approach will allow exploiting many of the already

  11. Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development.

    PubMed

    Hoane, Jessica S; Carruthers, Vernon B; Striepen, Boris; Morrison, David P; Entzeroth, Rolf; Howe, Daniel K

    2003-07-01

    Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the

  12. Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl–like molecules binding

    PubMed Central

    2013-01-01

    Background Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. Method We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Results Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. Conclusions The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins. PMID:23768251

  13. Analysis of Protein Phosphorylation and Its Functional Impact on Protein-Protein Interactions via Text Mining of the Scientific Literature.

    PubMed

    Wang, Qinghua; Ross, Karen E; Huang, Hongzhan; Ren, Jia; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

    2017-01-01

    Post-translational modifications (PTMs) are one of the main contributors to the diversity of proteoforms in the proteomic landscape. In particular, protein phosphorylation represents an essential regulatory mechanism that plays a role in many biological processes. Protein kinases, the enzymes catalyzing this reaction, are key participants in metabolic and signaling pathways. Their activation or inactivation dictate downstream events: what substrates are modified and their subsequent impact (e.g., activation state, localization, protein-protein interactions (PPIs)). The biomedical literature continues to be the main source of evidence for experimental information about protein phosphorylation. Automatic methods to bring together phosphorylation events and phosphorylation-dependent PPIs can help to summarize the current knowledge and to expose hidden connections. In this chapter, we demonstrate two text mining tools, RLIMS-P and eFIP, for the retrieval and extraction of kinase-substrate-site data and phosphorylation-dependent PPIs from the literature. These tools offer several advantages over a literature search in PubMed as their results are specific for phosphorylation. RLIMS-P and eFIP results can be sorted, organized, and viewed in multiple ways to answer relevant biological questions, and the protein mentions are linked to UniProt identifiers.

  14. Nano-LC-ESI MS/MS analysis of proteins in dried sea dragon Solenognathus hardwickii and bioinformatic analysis of its protein expression profiling.

    PubMed

    Zhang, Dong-Mei; Feng, Li-Xing; Li, Lu; Liu, Miao; Jiang, Bao-Hong; Yang, Min; Li, Guo-Qiang; Wu, Wan-Ying; Guo, De-An; Liu, Xuan

    2016-09-01

    The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry (nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and pI values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology (GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING (Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins.

  15. Bias tradeoffs in the creation and analysis of protein-protein interaction networks.

    PubMed

    Gillis, Jesse; Ballouz, Sara; Pavlidis, Paul

    2014-04-04

    Networks constructed from aggregated protein-protein interaction data are commonplace in biology. But the studies these data are derived from were conducted with their own hypotheses and foci. Focusing on data from budding yeast present in BioGRID, we determine that many of the downstream signals present in network data are significantly impacted by biases in the original data. We determine the degree to which selection bias in favor of biologically interesting bait proteins goes down with study size, while we also find that promiscuity in prey contributes more substantially in larger studies. We analyze interaction studies over time with respect to data in the Gene Ontology and find that reproducibly observed interactions are less likely to favor multifunctional proteins. We find that strong alignment between co-expression and protein-protein interaction data occurs only for extreme co-expression values, and use this data to suggest candidates for targets likely to reveal novel biology in follow-up studies. Protein-protein interaction data finds particularly heavy use in the interpretation of disease-causal variants. In principle, network data allows researchers to find novel commonalities among candidate genes. In this study, we detail several of the most salient biases contributing to aggregated protein-protein interaction databases. We find strong evidence for the role of selection and laboratory biases. Many of these effects contribute to the commonalities researchers find for disease genes. In order for characterization of disease genes and their interactions to not simply be an artifact of researcher preference, it is imperative to identify data biases explicitly. Based on this, we also suggest ways to move forward in producing candidates less influenced by prior knowledge. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes? Copyright © 2014 Elsevier B.V. All rights reserved.

  16. In silico analysis of candidate proteins sharing homology with Streptococcus agalactiae proteins and their role in male infertility.

    PubMed

    Parida, Rajeshwari; Samanta, Luna

    2017-02-01

    Leukocytospermia is a physiologic condition defined as human semen with a leukocyte count of >1 x 10(6) cells/ml that is often correlated with male infertility. Moreover, bacteriospermia has been associated with leukocytospermia ultimately leading to male infertility. We have found that semen samples with >1 x 10(6)/ml leukocytes and/or bacteriospermia have oxidative predominance as evidenced by augmented protein carbonyl and lipid peroxidation status of the semen which is implicated in sperm dysfunction. It has been reported that Streptococcus agalactiae is present in bacteriospermic samples. Previous research has shown that human leukocyte antigen beta chain paralog (HLA-DRB) alleles interact best with the infected sperm cells rather than the non-infected cells. Little is known about the interaction of major histocompatibility complex (MHC) present on leukocytes with the sperm upon bacterial infection and how it induces an immunological response which we have addressed by epitope mapping. Therefore, we examined MHC class II derived bacterial peptides which might have human sperm-related functional aspects. Twenty-two S. agalactiae proteins were obtained from PUBMED protein database for our study. Protein sequences with more than two accession numbers were aligned using CLUSTAL Omega to check their conservation pattern. Each protein sequence was then analyzed for T-cell epitope prediction against HLA-DRB alleles using the immune epitope database (IEDB) analysis tool. Out of a plethora of peptides obtained from this analysis, peptides corresponding to proteins of interest such as DNA binding response regulator, hyaluronate lyase and laminin binding protein were screened against the human proteome using Blastp. Interestingly, we have found bacterial peptides sharing homology with human peptides deciphering some of the important sperm functions. Antibodies raised against these probable bacterial antigens of fertility will not only help us understand the mechanism of

  17. Strain analysis of protein structures and low dimensionality of mechanical allosteric couplings.

    PubMed

    Mitchell, Michael R; Tlusty, Tsvi; Leibler, Stanislas

    2016-10-04

    In many proteins, especially allosteric proteins that communicate regulatory states from allosteric to active sites, structural deformations are functionally important. To understand these deformations, dynamical experiments are ideal but challenging. Using static structural information, although more limited than dynamical analysis, is much more accessible. Underused for protein analysis, strain is the natural quantity for studying local deformations. We calculate strain tensor fields for proteins deformed by ligands or thermal fluctuations using crystal and NMR structure ensembles. Strains-primarily shears-show deformations around binding sites. These deformations can be induced solely by ligand binding at distant allosteric sites. Shears reveal quasi-2D paths of mechanical coupling between allosteric and active sites that may constitute a widespread mechanism of allostery. We argue that strain-particularly shear-is the most appropriate quantity for analysis of local protein deformations. This analysis can reveal mechanical and biological properties of many proteins.

  18. Plant plasma membrane protein extraction and solubilization for proteomic analysis.

    PubMed

    Santoni, Véronique

    2007-01-01

    The plasma membrane (PM) exists as the interface between the cytosol and the environment in all living cells and is one of the most complex and differentiated membrane. The identification and characterization of membrane proteins (either extrinsic or intrinsic) is a crucial challenge since many of these proteins are involved in essential cellular functions such as cell signaling, osmoregulation, nutrition, and metabolism. Methods to isolate PM fractions vary according to organisms, tissues, and cell type. This chapter emphasizes isolation, from the model plant Arabidopsis thaliana, of PM fractions from a microsomal membrane fraction by two-phase partitioning, a methodology that utilizes the different surface properties of membranes. PM proteins that do not span the lipid bilayer are generally well recovered after 2D gel electrophoresis. By contrast, the recovery of transmembrane proteins requires first the depletion of the PM fraction from soluble proteins, being either cytosolic contaminants or functionally associated proteins, and second, to the use of specific solubilization procedures. This chapter presents protocols to strip PM based on alkaline treatment of membranes and to solubilize hydrophobic proteins to increase their recovery on 2D gels. Aquaporins that are highly hydrophobic proteins are used to probe the relevance of the procedures.

  19. NMR-based analysis of protein-ligand interactions.

    PubMed

    Cala, Olivier; Guillière, Florence; Krimm, Isabelle

    2014-02-01

    Physiological processes are mainly controlled by intermolecular recognition mechanisms involving protein-protein and protein-ligand (low molecular weight molecules) interactions. One of the most important tools for probing these interactions is high-field solution nuclear magnetic resonance (NMR) through protein-observed and ligand-observed experiments, where the protein receptor or the organic compounds are selectively detected. NMR binding experiments rely on comparison of NMR parameters of the free and bound states of the molecules. Ligand-observed methods are not limited by the protein molecular size and therefore have great applicability for analysing protein-ligand interactions. The use of these NMR techniques has considerably expanded in recent years, both in chemical biology and in drug discovery. We review here three major ligand-observed NMR methods that depend on the nuclear Overhauser effect-transferred nuclear Overhauser effect spectroscopy, saturation transfer difference spectroscopy and water-ligand interactions observed via gradient spectroscopy experiments-with the aim of reporting recent developments and applications for the characterization of protein-ligand complexes, including affinity measurements and structural determination.

  20. Systematic analysis of the twin cx(9)c protein family.

    PubMed

    Longen, Sebastian; Bien, Melanie; Bihlmaier, Karl; Kloeppel, Christine; Kauff, Frank; Hammermeister, Miriam; Westermann, Benedikt; Herrmann, Johannes M; Riemer, Jan

    2009-10-23

    The Mia40-Erv1 disulfide relay system is of high importance for mitochondrial biogenesis. Most so far identified substrates of this machinery contain either two cysteine-x(3)-cysteine (twin Cx(3)C) or two cysteine-x(9)-cysteine (twin Cx(9)C) motifs. While the first group is composed of well-characterized components of the mitochondrial import machinery, the molecular function of twin Cx(9)C proteins still remains unclear. To systematically characterize this protein family, we performed a database search to identify the full complement of Cx(9)C proteins in yeast. Thereby, we identified 14 potential family members, which, with one exception, are conserved among plants, fungi, and animals. Among these, three represent novel proteins, which we named Cmc2 to 4 (for Cx(9)C motif-containing protein) and which we demonstrated to be dependent for import on the Mia40-Erv1 disulfide relay. By testing deletion mutants of all 14 proteins for function of the respiratory chain, we found a critical function of most of these proteins for the assembly or stability of respiratory chain complexes. Our data suggest that already early during the evolution of eukaryotic cells, a multitude of twin Cx(9)C proteins developed, which exhibit largely nonredundant roles critical for the biogenesis of enzymes of the respiratory chain in mitochondria.

  1. Comparative Analysis of SWIRM Domain-Containing Proteins in Plants

    PubMed Central

    Gao, Yan; Yang, Songguang; Yuan, Lianyu; Cui, Yuhai; Wu, Keqiang

    2012-01-01

    Chromatin-remodeling complexes affect gene expression by using the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA. SWIRM (Swi3p, Rsc8p, and Moira) domain is an alpha-helical domain of about 85 residues in chromosomal proteins. SWIRM domain-containing proteins make up large multisubunit complexes by interacting with other chromatin modification factors and may have an important function in plants. However, little is known about SWIRM domain-containing proteins in plants. In this study, 67 SWIRM domain-containing proteins from 6 plant species were identified and analyzed. Plant SWIRM domain proteins can be divided into three distinct types: Swi-type, LSD1-type, and Ada2-type. Generally, the SWIRM domain forms a helix-turn-helix motif commonly found in DNA-binding proteins. The genes encoding SWIRM domain proteins in Oryza sativa are widely expressed, especially in pistils. In addition, OsCHB701 and OsHDMA701 were downregulated by cold stress, whereas OsHDMA701 and OsHDMA702 were significantly induced by heat stress. These observations indicate that SWIRM domain proteins may play an essential role in plant development and plant responses to environmental stress. PMID:22924025

  2. Proteomic analysis of cell surface proteins from Clostridium difficile.

    PubMed

    Wright, Anne; Wait, Robin; Begum, Shajna; Crossett, Ben; Nagy, Judit; Brown, Katherine; Fairweather, Neil

    2005-06-01

    Clostridium difficile is a bacterium that causes disease of the large intestine, particularly after treatment with antibiotics. The bacterium produces two toxins (A and B) that are responsible for the pathology of the disease. In addition, a number of bacterial virulence factors associated with adhesion to the gut have previously been identified, including the cell wall protein Cwp66, the high-molecular weight surface layer protein (HMW-SLP) and the flagella. As the genome sequence predicts many other cell wall associated proteins, we have investigated the diversity of proteins in cell wall extracts, with the aim of identifying further virulence factors. We have used a number of methods to remove the proteins associated with the cell wall of C. difficile. Two of the resulting extracts, obtained using low pH glycine treatment and lysozyme digestion of the cell wall, have been analysed in detail by two-dimensional electrophoresis and mass spectrometry. One hundred and nineteen spots, comprising 49 different proteins, have been identified. The two surface layer proteins (SLPs) are the most abundant proteins, and we have also found components of the flagellum. Interestingly, we have also determined that a number of paralogs of the HMW-SLP are expressed, and these could represent targets for further investigation as virulence factors.

  3. Fourier Analysis of Conservation Patterns in Protein Secondary Structure.

    PubMed

    Palaniappan, Ashok; Jakobsson, Eric

    2017-01-01

    Residue conservation is a common observation in alignments of protein families, underscoring positions important in protein structure and function. Though many methods measure the level of conservation of particular residue positions, currently we do not have a way to study spatial oscillations occurring in protein conservation patterns. It is known that hydrophobicity shows spatial oscillations in proteins, which is characterized by computing the hydrophobic moment of the protein domains. Here, we advance the study of moments of conservation of protein families to know whether there might exist spatial asymmetry in the conservation patterns of regular secondary structures. Analogous to the hydrophobic moment, the conservation moment is defined as the modulus of the Fourier transform of the conservation function of an alignment of related protein, where the conservation function is the vector of conservation values at each column of the alignment. The profile of the conservation moment is useful in ascertaining any periodicity of conservation, which might correlate with the period of the secondary structure. To demonstrate the concept, conservation in the family of potassium ion channel proteins was analyzed using moments. It was shown that the pore helix of the potassium channel showed oscillations in the moment of conservation matching the period of the α-helix. This implied that one side of the pore helix was evolutionarily conserved in contrast to its opposite side. In addition, the method of conservation moments correctly identified the disposition of the voltage sensor of voltage-gated potassium channels to form a 310 helix in the membrane.

  4. Aequorea green fluorescent protein analysis by flow cytometry

    SciTech Connect

    Ropp, J.D.; Cuthbertson, R.A.; Donahue, C.J.; Wolfgang-Kimball, D.

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types. The longer wavelength peak (470 nm) of GFP`s bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T- GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm. 29 refs., 5 figs.

  5. Aequorea green fluorescent protein analysis by flow cytometry.

    PubMed

    Ropp, J D; Donahue, C J; Wolfgang-Kimball, D; Hooley, J J; Chin, J Y; Hoffman, R A; Cuthbertson, R A; Bauer, K D

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263: 802-805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663-664, 1995) have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T-GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm.

  6. Feasibility study for the quantification of total protein content by multiple prompt gamma-ray analysis.

    PubMed

    Toh, Y; Murakami, Y; Furutaka, K; Kimura, A; Koizumi, M; Hara, K; Kin, T; Nakamura, S; Harada, H

    2012-06-01

    Protein is an important nutrient in foods. The classical nitrogen analysis method is the Kjeldahl technique, which is time-consuming and inconvenient. As a convenient method to quantify protein content in biological samples, the feasibility of application of multiple prompt gamma-ray analysis (MPGA) to the quantification was studied. Results for protein content are reported for several reference materials and prove the method to be reliable.

  7. Immunochemical and Biochemical Analysis of Adducts to DNA and Proteins After Exposure to Sulfur Mustard

    DTIC Science & Technology

    1993-05-13

    cQ.00o~33/o/ /9 IMMUNOCHEMICAL AND BIOCHEMICAL ANALYSIS OF O• ADDUCTS TO DNA AND PROTEINS AFTER EXPOSURE TO SULFUR MUSTARD _ == M G.P. van der Schans...chosen to define exposure to sulfur mustard (HD), based on immunochemical analysis of adducts of HD to DNA and proteins. These adducts are agent...calibration of the immunochemical assays. Analogous to HD-DNA adducts , reaction products with blood proteins may be used to establish HD exposure. Since it

  8. Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis.

    PubMed

    Kay, Richard; Barton, Chris; Ratcliffe, Lucy; Matharoo-Ball, Balwir; Brown, Pamela; Roberts, Jane; Teale, Phil; Creaser, Colin

    2008-10-01

    A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.

  9. Crysalis: an integrated server for computational analysis and design of protein crystallization.

    PubMed

    Wang, Huilin; Feng, Liubin; Zhang, Ziding; Webb, Geoffrey I; Lin, Donghai; Song, Jiangning

    2016-02-24

    The failure of multi-step experimental procedures to yield diffraction-quality crystals is a major bottleneck in protein structure determination. Accordingly, several bioinformatics methods have been successfully developed and employed to select crystallizable proteins. Unfortunately, the majority of existing in silico methods only allow the prediction of crystallization propensity, seldom enabling computational design of protein mutants that can be targeted for enhancing protein crystallizability. Here, we present Crysalis, an integrated crystallization analysis tool that builds on support-vector regression (SVR) models to facilitate computational protein crystallization prediction, analysis, and design. More specifically, the functionality of this new tool includes: (1) rapid selection of target crystallizable proteins at the proteome level, (2) identification of site non-optimality for protein crystallization and systematic analysis of all potential single-point mutations that might enhance protein crystallization propensity, and (3) annotation of target protein based on predicted structural properties. We applied the design mode of Crysalis to identify site non-optimality for protein crystallization on a proteome-scale, focusing on proteins currently classified as non-crystallizable. Our results revealed that site non-optimality is based on biases related to residues, predicted structures, physicochemical properties, and sequence loci, which provides in-depth understanding of the features influencing protein crystallization. Crysalis is freely available at http://nmrcen.xmu.edu.cn/crysalis/.

  10. Analysis of protein-protein interactions in the feline calicivirus replication complex.

    PubMed

    Kaiser, William J; Chaudhry, Yasmin; Sosnovtsev, Stanislav V; Goodfellow, Ian G

    2006-02-01

    Caliciviruses are a major cause of gastroenteritis in humans and cause a wide variety of other diseases in animals. Here, the characterization of protein-protein interactions between the individual proteins of Feline calicivirus (FCV), a model system for other members of the family Caliciviridae, is reported. Using the yeast two-hybrid system combined with a number of other approaches, it is demonstrated that the p32 protein (the picornavirus 2B analogue) of FCV interacts with p39 (2C), p30 (3A) and p76 (3CD). The FCV protease/RNA polymerase (ProPol) p76 was found to form homo-oligomers, as well as to interact with VPg and ORF2, the region encoding the major capsid protein VP1. A weak interaction was also observed between p76 and the minor capsid protein encoded by ORF3 (VP2). ORF2 protein was found to interact with VPg, p76 and VP2. The potential roles of the interactions in calicivirus replication are discussed.

  11. Protein haptenation by amoxicillin: high resolution mass spectrometry analysis and identification of target proteins in serum.

    PubMed

    Ariza, Adriana; Garzon, Davide; Abánades, Daniel R; de los Ríos, Vivian; Vistoli, Giulio; Torres, María J; Carini, Marina; Aldini, Giancarlo; Pérez-Sala, Dolores

    2012-12-21

    Allergy towards wide spectrum antibiotics such as amoxicillin (AX) is a major health problem. Protein haptenation by covalent conjugation of AX is considered a key process for the allergic response. However, the nature of the proteins involved has not been completely elucidated. Human serum albumin (HSA) is the most abundant protein in plasma and is considered a major target for haptenation by drugs, including β-lactam antibiotics. Here we report a procedure for immunological detection of AX-protein adducts with antibodies recognizing the lateral chain of the AX molecule. With this approach we detected human serum proteins modified by AX in vitro and identified HSA, transferrin and immunoglobulins heavy and light chains as prominent AX-modified proteins. Since HSA was the major AX target, we characterized AX-HSA interaction using high resolution LTQ orbitrap MS. At 0.5mg/mL AX, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. In molecular modeling studies Lys190 and Lys 199 were found the most reactive residues towards AX, with surrounding residues favoring adduct formation. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.

  12. A coevolution analysis for identifying protein-protein interactions by Fourier transform

    PubMed Central

    Yin, Changchuan; Yau, Stephen S. -T.

    2017-01-01

    Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI). PMID:28430779

  13. A coevolution analysis for identifying protein-protein interactions by Fourier transform.

    PubMed

    Yin, Changchuan; Yau, Stephen S-T

    2017-01-01

    Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI).

  14. Comparative analysis of protein-protein interactions in the defense response of rice and wheat.

    PubMed

    Cantu, Dario; Yang, Baoju; Ruan, Randy; Li, Kun; Menzo, Virginia; Fu, Daolin; Chern, Mawsheng; Ronald, Pamela C; Dubcovsky, Jorge

    2013-03-12

    Despite the importance of wheat as a major staple crop and the negative impact of diseases on its production worldwide, the genetic mechanisms and gene interactions involved in the resistance response in wheat are still poorly understood. The complete sequence of the rice genome has provided an extremely useful parallel road map for genetic and genomics studies in wheat. The recent construction of a defense response interactome in rice has the potential to further enhance the translation of advances in rice to wheat and other grasses. The objective of this study was to determine the degree of conservation in the protein-protein interactions in the rice and wheat defense response interactomes. As entry points we selected proteins that serve as key regulators of the rice defense response: the RAR1/SGT1/HSP90 protein complex, NPR1, XA21, and XB12 (XA21 interacting protein 12). Using available wheat sequence databases and phylogenetic analyses we identified and cloned the wheat orthologs of these four rice proteins, including recently duplicated paralogs, and their known direct interactors and tested 86 binary protein interactions using yeast-two-hybrid (Y2H) assays. All interactions between wheat proteins were further tested using in planta bimolecular fluorescence complementation (BiFC). Eighty three percent of the known rice interactions were confirmed when wheat proteins were tested with rice interactors and 76% were confirmed using wheat protein pairs. All interactions in the RAR1/SGT1/ HSP90, NPR1 and XB12 nodes were confirmed for the identified orthologous wheat proteins, whereas only forty four percent of the interactions were confirmed in the interactome node centered on XA21. We hypothesize that this reduction may be associated with a different sub-functionalization history of the multiple duplications that occurred in this gene family after the divergence of the wheat and rice lineages. The observed high conservation of interactions between proteins that

  15. Large-Scale Protein-Protein Interaction Analysis in Arabidopsis Mesophyll Protoplasts by Split Firefly Luciferase Complementation

    PubMed Central

    Li, Jian-Feng; Bush, Jenifer; Xiong, Yan; Li, Lei; McCormack, Matthew

    2011-01-01

    Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens. PMID:22096563

  16. Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges.

    PubMed

    Bush, Jeffrey; Makhatadze, George I

    2011-07-01

    It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ˜2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins.

  17. Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain.

    PubMed

    Kozielski, Frank; Riaz, Tahira; DeBonis, Salvatore; Koehler, Christian J; Kroening, Mario; Panse, Isabel; Strozynski, Margarita; Donaldson, Ian M; Thiede, Bernd

    2011-07-01

    The microtubule (MT) cytoskeleton is essential for a variety of cellular processes. MTs are finely regulated by distinct classes of MT-associated proteins (MAPs), which themselves bind to and are regulated by a large number of additional proteins. We have carried out proteome analyses of tubulin-rich and tubulin-depleted MAPs and their interacting partners isolated from bovine brain. In total, 573 proteins were identified giving us unprecedented access to brain-specific MT-associated proteins from mammalian brain. Most of the standard MAPs were identified and at least 500 proteins have been reported as being associated with MTs. We identified protein complexes with a large number of subunits such as brain-specific motor/adaptor/cargo complexes for kinesins, dynein, and dynactin, and proteins of an RNA-transporting granule. About 25% of the identified proteins were also found in the synaptic vesicle proteome. Analysis of the MS/MS data revealed many posttranslational modifications, amino acid changes, and alternative splice variants, particularly in tau, a key protein implicated in Alzheimer's disease. Bioinformatic analysis of known protein-protein interactions of the identified proteins indicated that the number of MAPs and their associated proteins is larger than previously anticipated and that our database will be a useful resource to identify novel binding partners.

  18. Cy5 total protein normalization in Western blot analysis.

    PubMed

    Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

    2015-10-01

    Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Comprehensive Analysis of Phosphorylated Proteins of E. coli Ribosomes

    PubMed Central

    Soung, George Y.; Miller, Jennifer L.; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  20. Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomes.

    PubMed

    Soung, George Y; Miller, Jennifer L; Koc, Hasan; Koc, Emine C

    2009-07-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in 24 Escherichia coli ribosomal proteins by tandem mass spectrometry. Detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining, and antibodies for phospho-Ser, Thr, and Tyr; or by mass spectrometry equipped with a capability to detect addition and loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, and L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given phosphorylation sites in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins.

  1. Comparative proteome analysis of egg yolk plasma proteins during storage.

    PubMed

    Gao, Dan; Qiu, Ning; Liu, Yaping; Ma, Meihu

    2017-06-01

    Physical changes such as chicken egg white thinning and egg yolk flattening occur during storage, implying a decline in egg quality. To reveal the deteriorative process related to chicken egg internal quality, a comparative proteomic method was used in this study to analyze the alterations in egg yolk plasma proteins at different storage times (0, 20 and 40 days) under an ambient temperature of 22 ± 2 °C. Using two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, 33 protein spots representing 12 proteins were identified with significant (P < 0.05) alterations in abundance at different storage times. The proteins that showed significant changes in abundance included serum albumin, vitellogenin fragments, IgY chains, ovalbumin, ovoinhibitor, α2 -macroglobulin-like protein 1-like, hemopexin, transthyretin, apolipoprotein A-I and β2 -glycoprotein I precursor. Accelerating degradation for most egg yolk plasma proteins was observed after prolonged storage (from day 20 to day 40). It is likely that the increased degradation of protease inhibitors such as ovoinhibitor and α2 -macroglobulin-like protein 1-like during prolonged storage lead to an imbalance of protease and antiprotease in egg yolk, which may play a key role in the degradation of egg yolk proteins. These findings will provide an insight into the effects of storage on egg yolk protein changes and give a deeper understanding of the deteriorative process of chicken egg yolk. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  2. Statistical analysis of interface similarity in crystals of homologous proteins.

    PubMed

    Xu, Qifang; Canutescu, Adrian A; Wang, Guoli; Shapovalov, Maxim; Obradovic, Zoran; Dunbrack, Roland L

    2008-08-29

    Many proteins function as homo-oligomers and are regulated via their oligomeric state. For some proteins, the stoichiometry of homo-oligomeric states under various conditions has been studied using gel filtration or analytical ultracentrifugation experiments. The interfaces involved in these assemblies may be identified using cross-linking and mass spectrometry, solution-state NMR, and other experiments. However, for most proteins, the actual interfaces that are involved in oligomerization are inferred from X-ray crystallographic structures using assumptions about interface surface areas and physical properties. Examination of interfaces across different Protein Data Bank (PDB) entries in a protein family reveals several important features. First, similarities in space group, asymmetric unit size, and cell dimensions and angles (within 1%) do not guarantee that two crystals are actually the same crystal form, containing similar relative orientations and interactions within the crystal. Conversely, two crystals in different space groups may be quite similar in terms of all the interfaces within each crystal. Second, NMR structures and an existing benchmark of PDB crystallographic entries consisting of 126 dimers as well as larger structures and 132 monomers were used to determine whether the existence or lack of common interfaces across multiple crystal forms can be used to predict whether a protein is an oligomer or not. Monomeric proteins tend to have common interfaces across only a minority of crystal forms, whereas higher-order structures exhibit common interfaces across a majority of available crystal forms. The data can be used to estimate the probability that an interface is biological if two or more crystal forms are available. Finally, the Protein Interfaces, Surfaces, and Assemblies (PISA) database available from the European Bioinformatics Institute is more consistent in identifying interfaces observed in many crystal forms compared with the PDB and the

  3. Human muscle proteins: analysis by two-dimensional electrophoresis

    SciTech Connect

    Giometti, C.S.; Danon, M.J.; Anderson, N.G.

    1983-09-01

    Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be cause by generalized muscle-fiber damage.

  4. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

    PubMed Central

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-01-01

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

  5. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.

    PubMed

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-05-24

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment.

  6. Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

    SciTech Connect

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K.; Schertler, Gebhard F. X.; Oprian, Daniel D.; Kern, Dorothee

    2015-12-24

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.

  7. Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

    DOE PAGES

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; ...

    2015-12-24

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsinmore » kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.« less

  8. Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

    DOE PAGES

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; ...

    2015-12-24

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsinmore » kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.« less

  9. Residual on column host cell protein analysis during lifetime studies of protein A chromatography.

    PubMed

    Lintern, Katherine; Pathak, Mili; Smales, C Mark; Howland, Kevin; Rathore, Anurag; Bracewell, Daniel G

    2016-08-26

    Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC-MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.

  10. Predicting protein subcellular locations with feature selection and analysis.

    PubMed

    Cai, Yudong; He, Jianfeng; Li, Xinlei; Feng, Kaiyan; Lu, Lin; Feng, Kairui; Kong, Xiangyin; Lu, Wencong

    2010-04-01

    In this paper, we propose a strategy to predict the subcellular locations of proteins by combining various feature selection methods. Firstly, proteins are coded by amino-acid composition and physicochemical properties, then these features are arranged by Minimum Redundancy Maximum Relevance method and further filtered by feature selection procedure. Nearest Neighbor Algorithm is used as a prediction model to predict the protein subcellular locations, and gains a correct prediction rate of 70.63%, evaluated by Jackknife cross-validation. Results of feature selection also enable us to identify the most important protein properties. The prediction software is available for public access on the website http://chemdata.shu.edu.cn/sub22/, which may play a important complementary role to a series of web-server predictors summarized recently in a review by Chou and Shen (Chou, K.C., Shen, H.B. Natural Science, 2009, 2, 63-92, http://www.scirp.org/journal/NS/).

  11. Determining protein function and interaction from genome analysis

    DOEpatents

    Eisenberg, David; Marcotte, Edward M.; Thompson, Michael J.; Pellegrini, Matteo; Yeates, Todd O.

    2004-08-03

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  12. A contact map matching approach to protein structure similarity analysis.

    PubMed

    de Melo, Raquel C; Lopes, Carlos Eduardo R; Fernandes, Fernando A; da Silveira, Carlos Henrique; Santoro, Marcelo M; Carceroni, Rodrigo L; Meira, Wagner; Araújo, Arnaldo de A

    2006-06-30

    We modeled the problem of identifying how close two proteins are structurally by measuring the dissimilarity of their contact maps. These contact maps are colored images, in which the chromatic information encodes the chemical nature of the contacts. We studied two conceptually distinct image-processing algorithms to measure the dissimilarity between these contact maps; one was a content-based image retrieval method, and the other was based on image registration. In experiments with contact maps constructed from the protein data bank, our approach was able to identify, with greater than 80% precision, instances of monomers of apolipoproteins, globins, plastocyanins, retinol binding proteins and thioredoxins, among the monomers of Protein Data Bank Select. The image registration approach was only slightly more accurate than the content-based image retrieval approach.

  13. Proteomic analysis of protein phosphatase Z1 from Candida albicans.

    PubMed

    Márkus, Bernadett; Szabó, Krisztina; Pfliegler, Walter P; Petrényi, Katalin; Boros, Enikő; Pócsi, István; Tőzsér, József; Csősz, Éva; Dombrádi, Viktor

    2017-01-01

    Protein phosphatase Z is a "novel type" fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for Ca

  14. Protein Structure Recognition: From Eigenvector Analysis to Structural Threading Method

    SciTech Connect

    Cao, Haibo

    2003-01-01

    In this work, they try to understand the protein folding problem using pair-wise hydrophobic interaction as the dominant interaction for the protein folding process. They found a strong correlation between amino acid sequences and the corresponding native structure of the protein. Some applications of this correlation were discussed in this dissertation include the domain partition and a new structural threading method as well as the performance of this method in the CASP5 competition. In the first part, they give a brief introduction to the protein folding problem. Some essential knowledge and progress from other research groups was discussed. This part includes discussions of interactions among amino acids residues, lattice HP model, and the design ability principle. In the second part, they try to establish the correlation between amino acid sequence and the corresponding native structure of the protein. This correlation was observed in the eigenvector study of protein contact matrix. They believe the correlation is universal, thus it can be used in automatic partition of protein structures into folding domains. In the third part, they discuss a threading method based on the correlation between amino acid sequences and ominant eigenvector of the structure contact-matrix. A mathematically straightforward iteration scheme provides a self-consistent optimum global sequence-structure alignment. The computational efficiency of this method makes it possible to search whole protein structure databases for structural homology without relying on sequence similarity. The sensitivity and specificity of this method is discussed, along with a case of blind test prediction. In the appendix, they list the overall performance of this threading method in CASP5 blind test in comparison with other existing approaches.

  15. Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants.

    PubMed

    Ling, Qihua; Jarvis, Paul

    2016-11-01

    Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with (35)S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

  16. Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins.

    PubMed

    Dunger, Germán; Garofalo, Cecilia G; Gottig, Natalia; Garavaglia, Betiana S; Rosa, María C Pereda; Farah, Chuck S; Orellano, Elena G; Ottado, Jorgelina

    2012-10-01

    Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  17. Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics

    PubMed Central

    Serwa, Remigiusz A.; Abaitua, Fernando; Krause, Eberhard; Tate, Edward W.; O’Hare, Peter

    2015-01-01

    Summary Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions. PMID:26256475

  18. Protein analysis by time-resolved measurements with an electro-switchable DNA chip

    PubMed Central

    Langer, Andreas; Hampel, Paul A.; Kaiser, Wolfgang; Knezevic, Jelena; Welte, Thomas; Villa, Valentina; Maruyama, Makiko; Svejda, Matej; Jähner, Simone; Fischer, Frank; Strasser, Ralf; Rant, Ulrich

    2013-01-01

    Measurements in stationary or mobile phases are fundamental principles in protein analysis. Although the immobilization of molecules on solid supports allows for the parallel analysis of interactions, properties like size or shape are usually inferred from the molecular mobility under the influence of external forces. However, as these principles are mutually exclusive, a comprehensive characterization of proteins usually involves a multi-step workflow. Here we show how these measurement modalities can be reconciled by tethering proteins to a surface via dynamically actuated nanolevers. Short DNA strands, which are switched by alternating electric fields, are employed as capture probes to bind target proteins. By swaying the proteins over nanometre amplitudes and comparing their motional dynamics to a theoretical model, the protein diameter can be quantified with Angström accuracy. Alterations in the tertiary protein structure (folding) and conformational changes are readily detected, and even post-translational modifications are revealed by time-resolved molecular dynamics measurements. PMID:23839273

  19. An investigation on the analytical potential of polymerized liposomes bound to lanthanide ions for protein analysis.

    PubMed

    Santos, Marina; Roy, Bidhan C; Goicoechea, Héctor; Campiglia, Andres D; Mallik, Sanku

    2004-09-01

    We present a promising approach to protein sensing based on Eu3+ ions incorporated into polymerized liposomes. The sensitization of Eu3+ is accomplished with 5-aminosalicylic acid, which provides energy transfer for a stable reference signal and a wide wavelength excitation range free from protein interference. The lipophilic character of polymerized liposomes provides the appropriate platform for protein interaction with the lanthanide ion. Quantitative analysis is based on the linear relationship between the luminescence signal of Eu3+ and protein concentration. Because no spectral shift of the lanthanide luminescence is observed upon protein interaction, qualitative analysis is based on the luminescence lifetime of polymerized liposomes. This parameter, which changes significantly upon protein-liposome interaction, follows a well-behaved single-exponential decay that might be useful for protein identification. Copyright 2004 American Chemical Society

  20. “Zero-length” Cross-linking in Solid State as an Approach for Analysis of Protein -Protein Interactions

    SciTech Connect

    Elshafey, Ahmed; Tolic, Nikola; Young, Malin M.; Sale, Kenneth L.; Smith, Richard D.; Kery, Vladimir

    2006-03-01

    Analyzing the architecture of protein complexes is a difficult task. Chemical cross-linking is often used in combination with mass spectrometric analysis to elucidate the interaction interfaces between proteins. We have developed a new approach for the analysis of interacting interfaces in protein complexes based on cross-linking in the solid state. Protein complexes are freeze-dried under vacuum and cross-links are introduced in the solid phase by dehydrating the protein in a non-water solvent, thus, creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross-linked proteins are digested into peptides with trypsin in both H216O and H218O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C-terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross-linked peptides.We demonstrated our method by cross-linking homooligomeric protein complexes alone or in a mixture of many other proteins. Cross-linking in the solid state was shown to be specific and reproducible. Glutathione-S-transferase (GST) from Schistosoma japonicum was studied in more detail. Twenty-seven unique intra-molecular and two inter-molecular cross-linked peptides were identified using tryptic mapping followed by LTQ-MS analysis. Identified cross-links were predominantly of amide origin, but six esters and thioesters were also found. Identified cross-linked peptides were validated by computational (visualization of cross-links in the three-dimensional [3D] structure of GST) and experimental (MS/MS) analyses. Most of the identified cross-links matched interacting peptides in the native 3D structure of GST indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru

  1. Target identification in Fusobacterium nucleatum by subtractive genomics approach and enrichment analysis of host-pathogen protein-protein interactions.

    PubMed

    Kumar, Amit; Thotakura, Pragna Lakshmi; Tiwary, Basant Kumar; Krishna, Ramadas

    2016-05-12

    Fusobacterium nucleatum, a well studied bacterium in periodontal diseases, appendicitis, gingivitis, osteomyelitis and pregnancy complications has recently gained attention due to its association with colorectal cancer (CRC) progression. Treatment with berberine was shown to reverse F. nucleatum-induced CRC progression in mice by balancing the growth of opportunistic pathogens in tumor microenvironment. Intestinal microbiota imbalance and the infections caused by F. nucleatum might be regulated by therapeutic intervention. Hence, we aimed to predict drug target proteins in F. nucleatum, through subtractive genomics approach and host-pathogen protein-protein interactions (HP-PPIs). We also carried out enrichment analysis of host interacting partners to hypothesize the possible mechanisms involved in CRC progression due to F. nucleatum. In subtractive genomics approach, the essential, virulence and resistance related proteins were retrieved from RefSeq proteome of F. nucleatum by searching against Database of Essential Genes (DEG), Virulence Factor Database (VFDB) and Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) tool respectively. A subsequent hierarchical screening to identify non-human homologous, metabolic pathway-independent/pathway-specific and druggable proteins resulted in eight pathway-independent and 27 pathway-specific druggable targets. Co-aggregation of F. nucleatum with host induces proinflammatory gene expression thereby potentiates tumorigenesis. Hence, proteins from IBDsite, a database for inflammatory bowel disease (IBD) research and those involved in colorectal adenocarcinoma as interpreted from The Cancer Genome Atlas (TCGA) were retrieved to predict drug targets based on HP-PPIs with F. nucleatum proteome. Prediction of HP-PPIs exhibited 186 interactions contributed by 103 host and 76 bacterial proteins. Bacterial interacting partners were accounted as putative targets. And enrichment analysis of host interacting partners showed statistically

  2. Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

    PubMed Central

    Ball, L. E.; Oatis, J. E.; Dharmasiri, K.; Busman, M.; Wang, J.; Cowden, L. B.; Galijatovic, A.; Chen, N.; Crouch, R. K.; Knapp, D. R.

    1998-01-01

    Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins. PMID:9541408

  3. Analysis of the proteins associated with platelet detergent resistant membranes.

    PubMed

    Szklanna, Paulina B; Foy, Martina; Wynne, Kieran; Byrne, Dwayne; Maguire, Patricia B

    2016-09-01

    Proteomic studies have facilitated the identification of proteins associated with the detergent-resistant membrane (DRM) fraction in a variety of cell types. Here, we have undertaken label-free quantitative (LFQ) proteomic profiling of the proteins associated with detergent-resistant plasma and internal membranes from resting and activated platelets. One hundred forty-one proteins were identified and raw data is available via ProteomeXchange with identifier PXD002554. The proteins identified include a myriad of important platelet signaling and trafficking proteins including Rap1b, Src, SNAP-23, syntaxin-11, and members of the previously unattributed Ragulator complex. Mean LFQ intensities calculated across three technical replicates for the three biological donors revealed that several important platelet signaling proteins altered their detergent solubility upon activation, including GPIbα, GPIbβ, Src, and 14-3-3ζ. Altered detergent solubility for GPIbα, following activation using a variety of platelet agonists, was confirmed by immunoblotting and further coimmunoprecipitation experiments revealed that GPIbα forms a complex with 14-3-3ζ that shifts into DRMs following activation. Taken together, proteomic profiling of platelet DRMs allowed greater insight in the complex biology of both DRMs and platelets and will be a useful subproteome to study platelet-related disease. All MS data have been deposited in the ProteomeXchange with identifier PXD002554 (http://proteomecentral.proteomexchange.org/dataset/PXD002554). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. A protein microarray-based analysis of S-nitrosylation

    PubMed Central

    Foster, Matthew W.; Forrester, Michael T.; Stamler, Jonathan S.

    2009-01-01

    The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of α-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols. PMID:19864628

  5. Identification, Analysis and Prediction of Protein Ubiquitination Sites

    PubMed Central

    Radivojac, Predrag; Vacic, Vladimir; Haynes, Chad; Cocklin, Ross R.; Mohan, Amrita; Heyen, Joshua W.; Goebl, Mark G.; Iakoucheva, Lilia M.

    2009-01-01

    Summary Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class-balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and proteins with very short half-lives were significantly enriched in the number of predicted ubiquitination sites. Proteome-wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory and cancer-associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease-associated mutations. UbPred is available at http://www.ubpred.org PMID:19722269

  6. An analysis of oligomerization interfaces in transmembrane proteins

    PubMed Central

    2013-01-01

    Background The amount of transmembrane protein (TM) structures solved to date is now large enough to attempt large scale analyses. In particular, extensive studies of oligomeric interfaces in the transmembrane region are now possible. Results We have compiled the first fully comprehensive set of validated transmembrane protein interfaces in order to study their features and assess what differentiates them from their soluble counterparts. Conclusions The general features of TM interfaces do not differ much from those of soluble proteins: they are large, tightly packed and possess many interface core residues. In our set, membrane lipids were not found to significantly mediate protein-protein interfaces. Although no G protein-coupled receptor (GPCR) was included in the validated set, we analyzed the crystallographic dimerization interfaces proposed in the literature. We found that the putative dimer interfaces proposed for class A GPCRs do not show the usual patterns of stable biological interfaces, neither in terms of evolution nor of packing, thus they likely correspond to crystal interfaces. We cannot however rule out the possibility that they constitute transient or weak interfaces. In contrast we do observe a clear signature of biological interface for the proposed dimer of the class F human Smoothened receptor. PMID:24134166

  7. Imaging and interferometric analysis of protein crystal growth

    NASA Astrophysics Data System (ADS)

    Raghunandan, Ranjini; Gupta, Anamika Sethia; Muralidhar, K.

    2008-04-01

    Protein crystals are grown under controlled temperature, concentration and vapor pressure conditions, usually by vapor diffusion, liquid-liquid diffusion and dialysis techniques. The present study examines the effects of protein concentration, drop size and reservoir height on the crystal growth of Hen Egg White Lysozyme (HEWL). Crystals are grown by the hanging drop vapor diffusion method using Modular VDX TM Plates. Due to the vapor pressure difference created between the protein drop and the reservoir, evaporation takes place till equilibrium is attained. Crystal formation takes place after a certain level of supersaturation is attained when the protein precipitates out in crystalline form. The observations revealed that the growth is faster for higher lysozyme concentration, smaller drop sizes and larger reservoir heights. The morphology of the crystals is viewed during the growth process using stereomicroscope. The number of crystals formed is the maximum for higher concentrations, drop sizes and reservoir heights. When the number of crystals formed is less, the size of the crystals is comparatively larger. The effect of evaporation of water vapor from the protein drop into the reservoir is studied using Mach-Zehnder interferometry. The recorded interferograms and shadowgraph images indicate the diffusion of condensed water into the reservoir. The radius of the drop is determined using the shadowgraph images of the growth process. The radius decreases with evaporation and the rate of decrease of radius is highest for higher protein concentrations, smaller drop sizes and larger reservoir heights.

  8. Comparative Analysis of Testis Protein Evolution in Rodents

    PubMed Central

    Turner, Leslie M.; Chuong, Edward B.; Hoekstra, Hopi E.

    2008-01-01

    Genes expressed in testes are critical to male reproductive success, affecting spermatogenesis, sperm competition, and sperm–egg interaction. Comparing the evolution of testis proteins at different taxonomic levels can reveal which genes and functional classes are targets of natural and sexual selection and whether the same genes are targets among taxa. Here we examine the evolution of testis-expressed proteins at different levels of divergence among three rodents, mouse (Mus musculus), rat (Rattus norvegicus), and deer mouse (Peromyscus maniculatus), to identify rapidly evolving genes. Comparison of expressed sequence tags (ESTs) from testes suggests that proteins with testis-specific expression evolve more rapidly on average than proteins with maximal expression in other tissues. Genes with the highest rates of evolution have a variety of functional roles including signal transduction, DNA binding, and egg–sperm interaction. Most of these rapidly evolving genes have not been identified previously as targets of selection in comparisons among more divergent mammals. To determine if these genes are evolving rapidly among closely related species, we sequenced 11 of these genes in six Peromyscus species and found evidence for positive selection in five of them. Together, these results demonstrate rapid evolution of functionally diverse testis-expressed proteins in rodents, including the identification of amino acids under lineage-specific selection in Peromyscus. Evidence for positive selection among closely related species suggests that changes in these proteins may have consequences for reproductive isolation. PMID:18689890

  9. A protein microarray-based analysis of S-nitrosylation.

    PubMed

    Foster, Matthew W; Forrester, Michael T; Stamler, Jonathan S

    2009-11-10

    The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols.

  10. Microfluidics for the analysis of membrane proteins: how do we get there?

    PubMed

    Battle, Katrina N; Uba, Franklin I; Soper, Steven A

    2014-08-01

    The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples such as serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to the cytosolic proteins, have various functions and can provide insight into the state of a disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques, which are not always suitable for particular protein samples such as membrane proteins. Microfluidics offers the potential as a fully automated platform for the efficient and high-throughput analysis of complex samples, such as membrane proteins, and do so with performance metrics that exceed their bench-top counterparts. In recent years, there have been various improvements to microfluidics and their use for proteomic analysis as reported in the literature. Consequently, this review presents an overview of the traditional proteomic-processing pipelines for membrane proteins and insights into new technological developments with a focus on the applicability of microfluidics for the analysis of membrane proteins. Sample preparation techniques will be discussed in detail and novel interfacing strategies as it relates to MS will be highlighted. Lastly, some general conclusions and future perspectives are presented.

  11. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

    DOE PAGES

    Scavuzzo-Duggan, Tess R.; Chaves, Arielle M.; Roberts, Alison W.

    2015-07-14

    Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Westernmore » blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.« less

  12. Escherichia coli as host for membrane protein structure determination: a global analysis

    PubMed Central

    Hattab, Georges; Warschawski, Dror E.; Moncoq, Karine; Miroux, Bruno

    2015-01-01

    The structural biology of membrane proteins (MP) is hampered by the difficulty in producing and purifying them. A comprehensive analysis of protein databases revealed that 213 unique membrane protein structures have been obtained after production of the target protein in E. coli. The primary expression system used was the one based on the T7 RNA polymerase, followed by the arabinose and T5 promoter based expression systems. The C41λ(DE3) and C43λ(DE3) bacterial mutant hosts have contributed to 28% of non E. coli membrane protein structures. A large scale analysis of expression protocols demonstrated a preference for a combination of bacterial host-vector together with a bimodal distribution of induction temperature and of inducer concentration. Altogether our analysis provides a set of rules for the optimal use of bacterial expression systems in membrane protein production. PMID:26160693

  13. In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells.

    PubMed

    Espino, Jessica A; Mali, Vishaal S; Jones, Lisa M

    2015-08-04

    Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.

  14. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo

    PubMed Central

    Hostettler, Lola; Grundy, Laura; Käser-Pébernard, Stéphanie; Wicky, Chantal; Schafer, William R.; Glauser, Dominique A.

    2017-01-01

    The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro. The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans—a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3–5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression. PMID:28108553

  15. Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins.

    PubMed

    Deatherage Kaiser, Brooke L; Wunschel, David S; Sydor, Michael A; Warner, Marvin G; Wahl, Karen L; Hutchison, Janine R

    2015-11-01

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied.

  16. Two-dimensional gel-based protein standardization verified by western blot analysis.

    PubMed

    Haniu, Hisao; Watanabe, Daisuke; Kawashima, Yusuke; Matsumoto, Hiroyuki

    2015-01-01

    In data presentation of biochemical investigation the amount of a target protein is shown in the y-axis against the x-axis representing time, concentrations of various agents, or other parameters. Western blot is a versatile and convenient tool in such an analysis to quantify and display the amount of proteins. In western blot, so-called housekeeping gene product(s), or "housekeeping proteins," are widely used as internal standards. The rationale of using housekeeping proteins for standardization of western blot is based on the assumption that the expression of chosen housekeeping gene is always constant, which could be false under certain physiological or pathological conditions. We have devised a two-dimensional gel-based standardization method in which the protein content of each sample is determined by scanning the total protein density of two-dimensional gels and the expression of each protein is quantified as the density ratio of each protein divided by the density of the total proteins on the two-dimensional gel. The advantage of this standardization method is that it is not based on any presumed "housekeeping proteins" that are supposed to be being expressed constantly under all physiological conditions. We will show that the total density of a two-dimensional gel can render a reliable protein standardization parameter by running western blot analysis on one of the proteins analyzed by two-dimensional gels.

  17. Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation.

    PubMed

    Eitzinger, Nicole; Wagner, Volker; Weisheit, Wolfram; Geimer, Stefan; Boness, David; Kreimer, Georg; Mittag, Maria

    2015-01-01

    Flagellate green algae possess a visual system, the eyespot. In Chlamydomonas reinhardtii it is situated at the edge of the chloroplast and consists of two carotenoid rich lipid globule layers subtended by thylakoid membranes (TM) that are attached to both chloroplast envelope membranes and a specialized area of the plasma membrane (PM). A former analysis of an eyespot fraction identified 203 proteins. To increase the understanding of eyespot related processes, knowledge of the protein composition of the membranes in its close vicinity is desirable. Here, we present a purification procedure that allows isolation of intact eyespots. This gain in intactness goes, however, hand in hand with an increase of contaminants from other organelles. Proteomic analysis identified 742 proteins. Novel candidates include proteins for eyespot development, retina-related proteins, ion pumps, and membrane-associated proteins, calcium sensing proteins as well as kinases, phosphatases and 14-3-3 proteins. Methylation of proteins at Arg or Lys is known as an important posttranslational modification involved in, e.g., signal transduction. Here, we identify several proteins from eyespot fractions that are methylated at Arg and/or Lys. Among them is the eyespot specific SOUL3 protein that influences the size and position of the eyespot and EYE2, a protein important for its development.

  18. Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation

    PubMed Central

    Eitzinger, Nicole; Wagner, Volker; Weisheit, Wolfram; Geimer, Stefan; Boness, David; Kreimer, Georg; Mittag, Maria

    2015-01-01

    Flagellate green algae possess a visual system, the eyespot. In Chlamydomonas reinhardtii it is situated at the edge of the chloroplast and consists of two carotenoid rich lipid globule layers subtended by thylakoid membranes (TM) that are attached to both chloroplast envelope membranes and a specialized area of the plasma membrane (PM). A former analysis of an eyespot fraction identified 203 proteins. To increase the understanding of eyespot related processes, knowledge of the protein composition of the membranes in its close vicinity is desirable. Here, we present a purification procedure that allows isolation of intact eyespots. This gain in intactness goes, however, hand in hand with an increase of contaminants from other organelles. Proteomic analysis identified 742 proteins. Novel candidates include proteins for eyespot development, retina-related proteins, ion pumps, and membrane-associated proteins, calcium sensing proteins as well as kinases, phosphatases and 14-3-3 proteins. Methylation of proteins at Arg or Lys is known as an important posttranslational modification involved in, e.g., signal transduction. Here, we identify several proteins from eyespot fractions that are methylated at Arg and/or Lys. Among them is the eyespot specific SOUL3 protein that influences the size and position of the eyespot and EYE2, a protein important for its development. PMID:26697039

  19. A Bayesian framework for cell-level protein network analysis for multivariate proteomics image data

    NASA Astrophysics Data System (ADS)

    Kovacheva, Violet N.; Sirinukunwattana, Korsuk; Rajpoot, Nasir M.

    2014-03-01

    The recent development of multivariate imaging techniques, such as the Toponome Imaging System (TIS), has facilitated the analysis of multiple co-localisation of proteins. This could hold the key to understanding complex phenomena such as protein-protein interaction in cancer. In this paper, we propose a Bayesian framework for cell level network analysis allowing the identification of several protein pairs having significantly higher co-expression levels in cancerous tissue samples when compared to normal colon tissue. It involves segmenting the DAPI-labeled image into cells and determining the cell phenotypes according to their protein-protein dependence profile. The cells are phenotyped using Gaussian Bayesian hierarchical clustering (GBHC) after feature selection is performed. The phenotypes are then analysed using Difference in Sums of Weighted cO-dependence Profiles (DiSWOP), which detects differences in the co-expression patterns of protein pairs. We demonstrate that the pairs highlighted by the proposed framework have high concordance with recent results using a different phenotyping method. This demonstrates that the results are independent of the clustering method used. In addition, the highlighted protein pairs are further analysed via protein interaction pathway databases and by considering the localization of high protein-protein dependence within individual samples. This suggests that the proposed approach could identify potentially functional protein complexes active in cancer progression and cell differentiation.

  20. Atomic Analysis of Protein-Protein Interfaces with Known Inhibitors: The 2P2I Database

    PubMed Central

    Bourgeas, Raphaël; Basse, Marie-Jeanne

    2010-01-01

    Background In the last decade, the inhibition of protein-protein interactions (PPIs) has emerged from both academic and private research as a new way to modulate the activity of proteins. Inhibitors of these original interactions are certainly the next generation of highly innovative drugs that will reach the market in the next decade. However, in silico design of such compounds still remains challenging. Methodology/Principal Findings Here we describe this particular PPI chemical space through the presentation of 2P2IDB, a hand-curated database dedicated to the structure of PPIs with known inhibitors. We have analyzed protein/protein and protein/inhibitor interfaces in terms of geometrical parameters, atom and residue properties, buried accessible surface area and other biophysical parameters. The interfaces found in 2P2IDB were then compared to those of representative datasets of heterodimeric complexes. We propose a new classification of PPIs with known inhibitors into two classes depending on the number of segments present at the interface and corresponding to either a single secondary structure element or to a more globular interacting domain. 2P2IDB complexes share global shape properties with standard transient heterodimer complexes, but their accessible surface areas are significantly smaller. No major conformational changes are seen between the different states of the proteins. The interfaces are more hydrophobic than general PPI's interfaces, with less charged residues and more non-polar atoms. Finally, fifty percent of the complexes in the 2P2IDB dataset possess more hydrogen bonds than typical protein-protein complexes. Potential areas of study for the future are proposed, which include a new classification system consisting of specific families and the identification of PPI targets with high druggability potential based on key descriptors of the interaction. Conclusions 2P2I database stores structural information about PPIs with known inhibitors and

  1. Microwave-assisted protein solubilization for mass spectrometry-based shotgun proteome analysis.

    PubMed

    Ye, Xiaoxia; Li, Liang

    2012-07-17

    Protein solubilization is a key step in mass spectrometry-based shotgun proteome analysis. We describe a microwave-assisted protein solubilization (MAPS) method to dissolve proteins in reagents, such as NH(4)HCO(3) and urea, with high efficiency and with an added benefit that the solubilized proteins are denatured to become more susceptible to trypsin digestion, compared to other conventional protein solubilization techniques. In this method, a sample vial containing proteins suspended in a solubilization reagent is placed inside a domestic microwave oven and subjected to microwave irradiation for 30 s, followed by cooling the sample on ice to room temperature (~40 s) and then intermittent homogenization by vortex for 2 min. This cycle of microwave irradiation, cooling, and homogenization is repeated six times. In this way, sample overheating can be avoided, and a maximum amount of protein can be dissolved. It was shown that in the case of trypsin digestion of bovine serum albumen (BSA) more peptides and higher sequence coverage could be obtained from the protein dissolved by the MAPS method than the conventional heating, sonication, or vortex method. Compared to the most commonly used vortex-assisted protein solubilization method, MAPS reduces the solubilization time significantly, increases the amount of protein dissolvable in a reagent, and increases the number of proteins and peptides identified from a proteome sample. For example, in the proteome analysis of an Escherichia coli K-12 integral membrane protein extract, the MAPS method in combination with sequential protein solubilization and shotgun two-dimensional liquid chromatography tandem mass spectrometry analysis identified a total of 1291 distinct proteins and 10363 peptides, compared to 1057 proteins and 6261 peptides identified using the vortex method. Because MAPS can be done using an inexpensive microwave oven, this method can be readily adopted.

  2. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  3. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering.

    PubMed

    Blackburn, Matthew C; Petrova, Ekaterina; Correia, Bruno E; Maerkl, Sebastian J

    2016-04-20

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF-DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.

  4. In Silico Analysis of Tumor Necrosis Factor α-Induced Protein 8-Like-1 (TIPE1) Protein

    PubMed Central

    Su, Zhaoliang; Wang, Shengjun; Xu, Huaxi

    2015-01-01

    Tumor necrosis factor α-induced protein 8 (TNFAIP8)-like protein 1 (TIPE1) was a member of TNFAIP8 family. Previous studies have shown that TIPE1 could induce apoptosis in hepatocellular carcinoma. In this study, we attempted to predict its potential structure. Bioinformatic analysis of TIPE1 was performed to predict its potential structure using the bioinfomatic web services or softwares. The results showed that the amino acid sequences of TIPE1 were well conserved in mammals. No signal peptide and no transmembrane domain existed in human TIPE1. The aliphatic index of TIPE1 was 100.75 and the theoretical pI was 9.57. TIPE1 was a kind of stable protein and its grand average of hydropathicity was -0.108. Various post-translational modifications were also speculated to exist in TIPE1. In addition, the results of Swiss-Model Server and Swiss-Pdb Viewer program revealed that the predicted three-dimensional structure of TIPE1 protein was stable and it may accord with the rule of stereochemistry. TIPE1 was predicted to interact with FBXW5, caspase8 and so on. In conclusion, TIPE1 may be a stable protein with no signal peptide and no transmembrane domain. The bioinformatic analysis of TIPE1 will provide the basis for the further study on the function of TIPE1. PMID:26207809

  5. Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition

    NASA Astrophysics Data System (ADS)

    Winzen, S.; Schoettler, S.; Baier, G.; Rosenauer, C.; Mailaender, V.; Landfester, K.; Mohr, K.

    2015-02-01

    Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A

  6. A genome-wide resource for the analysis of protein localisation in Drosophila.

    PubMed

    Sarov, Mihail; Barz, Christiane; Jambor, Helena; Hein, Marco Y; Schmied, Christopher; Suchold, Dana; Stender, Bettina; Janosch, Stephan; K J, Vinay Vikas; Krishnan, R T; Krishnamoorthy, Aishwarya; Ferreira, Irene R S; Ejsmont, Radoslaw K; Finkl, Katja; Hasse, Susanne; Kämpfer, Philipp; Plewka, Nicole; Vinis, Elisabeth; Schloissnig, Siegfried; Knust, Elisabeth; Hartenstein, Volker; Mann, Matthias; Ramaswami, Mani; VijayRaghavan, K; Tomancak, Pavel; Schnorrer, Frank

    2016-02-20

    The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.

  7. Description and recognition of regular and distorted secondary structures in proteins using the automated protein structure analysis method.

    PubMed

    Ranganathan, Sushilee; Izotov, Dmitry; Kraka, Elfi; Cremer, Dieter

    2009-08-01

    The Automated Protein Structure Analysis (APSA) method, which describes the protein backbone as a smooth line in three-dimensional space and characterizes it by curvature kappa and torsion tau as a function of arc length s, was applied on 77 proteins to determine all secondary structural units via specific kappa(s) and tau(s) patterns. A total of 533 alpha-helices and 644 beta-strands were recognized by APSA, whereas DSSP gives 536 and 651 units, respectively. Kinks and distortions were quantified and the boundaries (entry and exit) of secondary structures were classified. Similarity between proteins can be easily quantified using APSA, as was demonstrated for the roll architecture of proteins ubiquitin and spinach ferridoxin. A twenty-by-twenty comparison of all alpha domains showed that the curvature-torsion patterns generated by APSA provide an accurate and meaningful similarity measurement for secondary, super secondary, and tertiary protein structure. APSA is shown to accurately reflect the conformation of the backbone effectively reducing three-dimensional structure information to two-dimensional representations that are easy to interpret and understand. 2008 Wiley-Liss, Inc.

  8. Analysis of proteins with the 'hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

    PubMed Central

    Pidugu, Lakshmi S; Maity, Koustav; Ramaswamy, Karthikeyan; Surolia, Namita; Suguna, Kaza

    2009-01-01

    Background The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. Results We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. Conclusion The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects. PMID:19473548

  9. Analysis of incomplete gene expression dataset through protein-protein interaction information.

    PubMed

    Massanet-Vila, Raimon; Padró, Teresa; Cardús, Anna; Badimon, Lina; Caminal, Pere; Perera, Alexandre

    2011-01-01

    This paper shows a graph based method to analyze proteomic expression data. The method allows the prediction of the expression of genes not measured by the gene expression technology based on the local connectivity properties of the measured differentially expressed gene set. The prediction of the expression jointly with the stability of this prediction as a function of the variation of the initial expressed set is computed. The method is able to correctly predict one third of the proteins with independence of variations on the selection of the initial set. The algorithm is validated through a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF) protein expression experiment aiming the study of the protein expression patterns and post-translational modifications in human endothelial vascular cells exposed to atherosclerotic levels of Low Density Lipoproteins (LDL).

  10. Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in live cells.

    PubMed

    Kerppola, Tom K

    2013-08-01

    Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions and modifications in living cells. It is based on the facilitated association of two nonfluorescent fragments of a fluorescent protein fused to putative interaction partners. The intrinsic fluorescence of the active complex enables detection of protein interactions with high sensitivity, fine spatial resolution, and minimal perturbation of the cells. This protocol outlines methods to analyze protein interactions in cultured mammalian cells using BiFC, but can be readily adapted to any cell type or aerobically grown organism that can be genetically modified to express the fusion proteins.

  11. Structural and functional analysis of fatty acid-binding proteins

    PubMed Central

    Storch, Judith; McDermott, Lindsay

    2009-01-01

    The mammalian FA-binding proteins (FABPs) bind long-chain FA with high affinity. The large number of FABP types is suggestive of distinct functions in specific tissues. Multiple experimental approaches have shown that individual FABPs possess both unique and overlapping functions, some of which are based on specific elements in the protein structure. Although FA binding affinities for all FABPs tend to correlate directly with FA hydrophobicity, structure-function studies indicate that subtle three-dimensional changes that occur upon ligand binding may promote specific protein-protein or protein-membrane interactions that ultimately determine the function of each FABP. The conformational changes are focused in the FABP helical/portal domain, a region that was identified by in vitro studies to be vital for the FA transport properties of the FABPs. Thus, the FABPs modulate intracellular lipid homeostasis by regulating FA transport in the nuclear and extra-nuclear compartments of the cell; in so doing, they also impact systemic energy homeostasis. PMID:19017610

  12. Identification of human olfactory cleft mucus proteins using proteomic analysis.

    PubMed

    Débat, Hélène; Eloit, Corinne; Blon, Florence; Sarazin, Benoît; Henry, Céline; Huet, Jean-Claude; Trotier, Didier; Pernollet, Jean-Claude

    2007-05-01

    In humans, the olfactory epithelium is located in two narrow passages, the olfactory clefts, at the upper part of the nasal cavities. The olfactory epithelium is covered by a mucus layer which is essential for the function of the olfactory neurons that are directly connected with the brain through the cribriform plate. This anatomical weakness of the brain protection may be the source of infection. Little is known about the composition of this mucus in humans. Previous proteomic analyses have been performed on washes of the entire nasal cavities and therefore might better correspond to the mucus over the respiratory epithelium than to the mucus covering the olfactory epithelium. In the present study, we sampled the olfactory mucus directly from the clefts of 16 healthy adult volunteers, and 83 proteins were identified in the samples using two-dimensional gel electrophoresis, MALDI-TOF, RPLC, and Edman sequencing. Forty-three proteins were not previously observed either in nasal mucus sampled through washings, saliva, tear, or cerebrospinal fluid. In Accordance with the data in the protein databases, the most abundant proteins are secreted, whereas some others correspond to intracellular proteins covering a large range of functions: anti-inflammatory, antimicrobial, protease inhibition, antioxidant, transport, transcription, transduction, cytoskeletal, regulation, binding, and metabolism of odorant molecules. This study clearly demonstrates the complexity of the mucus covering the human olfactory epithelium, which might comprise potential markers for characterizing pathophysiological states.

  13. Expression analysis of BRUCE protein in esophageal squamous cell carcinoma.

    PubMed

    Salehi, Somayeh; Jafarian, Amir Hossein; Forghanifard, Mohammad Mahdi

    2016-10-01

    Apoptosis is a form of cell death in response to diverse stressful physiological or pathological stimuli. One of the most important gene families involved in apoptosis is inhibitors of apoptosis. As a member of inhibitors of apoptosis, BRUCE can suppress apoptosis and promote cell division. Because esophageal squamous cell carcinoma (ESCC) cells, as well as other cancer cells, are immortal, our aim in this study was to analyze BRUCE protein expression in ESCC and evaluate its correlation with tumoral clinicopathologic features. Fifty ESCC specimens were examined for BRUCE protein expression using immunohistochemistry. A defined scoring method was applied. BRUCE protein was detected in 82% of tumors. Tumor progression stage and invasion depth correlated significantly with BRUCE protein expression (P=.019 and .005, respectively). Furthermore, association of BRUCE expression with tumor location was near significant (P=.058). The correlation of BRUCE overexpression in ESCC and disease aggressiveness may confirm the importance of BRUCE in ESCC progression and invasiveness. Therefore, BRUCE protein may be a molecular marker for aggressive ESCC and, thus, a potential therapeutic target to inhibit tumor cell progression and invasion. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Purification and Structural Analysis of LEM-Domain Proteins.

    PubMed

    Herrada, Isaline; Bourgeois, Benjamin; Samson, Camille; Buendia, Brigitte; Worman, Howard J; Zinn-Justin, Sophie

    2016-01-01

    LAP2-emerin-MAN1 (LEM)-domain proteins are modular proteins characterized by the presence of a conserved motif of about 50 residues. Most LEM-domain proteins localize at the inner nuclear membrane, but some are also found in the endoplasmic reticulum or nuclear interior. Their architecture has been analyzed by predicting the limits of their globular domains, determining the 3D structure of these domains and in a few cases calculating the 3D structure of specific domains bound to biological targets. The LEM domain adopts an α-helical fold also found in SAP and HeH domains of prokaryotes and unicellular eukaryotes. The LEM domain binds to BAF (barrier-to-autointegration factor; BANF1), which interacts with DNA and tethers chromatin to the nuclear envelope. LAP2 isoforms also share an N-terminal LEM-like domain, which binds DNA. The structure and function of other globular domains that distinguish LEM-domain proteins from each other have been characterized, including the C-terminal dimerization domain of LAP2α and C-terminal WH and UHM domains of MAN1. LEM-domain proteins also have large intrinsically disordered regions that are involved in intra- and intermolecular interactions and are highly regulated by posttranslational modifications in vivo.

  15. Proteomic analysis of Col11a1-associated protein complexes

    PubMed Central

    Brown, Raquel J.; Mallory, Christopher; McDougal, Owen M.; Oxford, Julia Thom

    2012-01-01

    Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen alpha 1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen alpha 1(XI). Mass spectrometry was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen alpha 1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration. PMID:22038862

  16. Protein Structural Analysis via Mass Spectrometry-Based Proteomics

    PubMed Central

    Artigues, Antonio; Nadeau, Owen W.; Rimmer, Mary Ashley; Villar, Maria T.; Du, Xiuxia; Fenton, Aron W.; Carlson, Gerald M.

    2017-01-01

    Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: 1) hydrogen/deuterium exchange (HDX), 2) limited proteolysis, and 3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides. Structural information on surface exposed, flexible or disordered linker regions of proteins can be achieved through limited proteolysis, using a variety of proteases and only small extents of digestion. CX refers to the covalent coupling of distinct chemical species and has been used to analyze the structure, function and interactions of proteins by identifying crosslinking sites that are formed by small multi-functional reagents, termed crosslinkers. Each of these MS applications is capable of revealing structural information for proteins when used either with or without other typical high resolution techniques, including NMR and X-ray crystallography. PMID:27975228

  17. RNA-protein analysis using a conditional CRISPR nuclease.

    PubMed

    Lee, Ho Young; Haurwitz, Rachel E; Apffel, Alex; Zhou, Kaihong; Smart, Brian; Wenger, Craig D; Laderman, Stephen; Bruhn, Laurakay; Doudna, Jennifer A

    2013-04-02

    RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5' ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.

  18. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  19. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  20. Protein

    USDA-ARS?s Scientific Manuscript database

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  1. Analysis of O2-binding Sites in Proteins Using Gas-Pressure NMR Spectroscopy: Outer Surface Protein A.

    PubMed

    Kawamura, Takahiro; Wakamoto, Takuro; Kitazawa, Soichiro; Sakuraba, Shun; Kameda, Tomoshi; Kitahara, Ryo

    2017-05-09

    Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O2-binding sites by O2-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer β-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O2-induced PREs for amide protons located around the largest cavity and at the central β-sheet. We suggested three potential O2-accessible sites in the protein based on the 1/r(6) distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central β-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O2 generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O2 within the largest cavity, egress of O2 from the cavity, and ingress of O2 in the surface crevice of the β-sheet. These molecular dynamics simulation results qualitatively explained the O2-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Characterization and identification of a novel candidate vaccine protein through systematic analysis of extracellular proteins of Erysipelothrix rhusiopathiae.

    PubMed

    Shi, Fang; Ogawa, Yohsuke; Sano, Akiyuki; Harada, Tomoyuki; Hirota, Jiro; Eguchi, Masahiro; Oishi, Eiji; Shimoji, Yoshihiro

    2013-12-01

    Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

  3. Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

    PubMed Central

    Shi, Fang; Ogawa, Yohsuke; Sano, Akiyuki; Harada, Tomoyuki; Hirota, Jiro; Eguchi, Masahiro; Oishi, Eiji

    2013-01-01

    Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen. PMID:24019408

  4. Chromosome protein framework from proteome analysis of isolated human metaphase chromosomes.

    PubMed

    Fukui, Kiichi; Uchiyama, Susumu

    2007-01-01

    We have presented a structural model of the chromosome based on its constituent proteins. Development of a method of mass isolation for intact human metaphase chromosomes and proteome analysis by mass spectrometry of the isolated chromosomal proteins enabled us to develop a four-layer structural model of human metaphase chromosomes. The model consists of four layers, each with different chromosomal protein sets, i.e., chromosome coating proteins (CCPs), chromosome peripheral proteins (CPPs), chromosome structural proteins (CSPs), and chromosome fibrous proteins (CFPs). More than 200 identified proteins have been classified and assigned to the four layers with each layer occupying a distinct region of the chromosome. CCPs are localized at the most outer regions of the chromosomes and they attach to the regions tentatively and occasionally. CCPs include mostly mitochondrial and cytoplasmic proteins, e.g., 70 kDa heat shock protein 9B and Hsp60. CPPs are also localized at the peripheral regions of the chromosomes, but as the essential part of the chromosomes. CPPs include nucleolin, lamin A/C, fibrillarin, etc. CSPs are the primary chromosomal structure proteins, and include topoisomerase IIalpha, condensin subunits, histones, etc. CFPs have a fibrous nature, e.g., beta-actin, vimentin, myosin II, tublin, etc. A data set of these proteins, which we developed, contains essential chromosome proteins with classified information based on this four-layer model and presents useful leads for further studies on chromosomal structure and function.

  5. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  6. A dielectrophoresis-impedance method for protein detection and analysis

    NASA Astrophysics Data System (ADS)

    Mohamad, Ahmad Sabry; Hamzah, Roszymah; Hoettges, Kai F.; Hughes, Michael Pycraft

    2017-01-01

    Dielectrophoresis (DEP) has increasingly been used for the assessment of the electrical properties of molecular scale objects including proteins, DNA, nanotubes and nanowires. However, whilst techniques have been developed for the electrical characterisation of frequency-dependent DEP response, biomolecular study is usually limited to observation using fluorescent markers, limiting its applicability as a characterisation tool. In this paper we present a label-free, impedance-based method of characterisation applied to the determination of the electrical properties of colloidal protein molecules, specifically Bovine Serum Albumin (BSA). By monitoring the impedance between electrodes as proteins collect, it is shown to be possible to observe multi-dispersion behaviour. A DEP dispersion exhibited at 400 kHz is attributable to the orientational dispersion of the molecule, whilst a second, higher-frequency dispersion is attributed to a Maxwell-Wagner type dispersion; changes in behaviour with medium conductivity suggest that this is strongly influenced by the electrical double layer surrounding the molecule.

  7. Protein solution photomodification analysis by means of craquelure structures

    NASA Astrophysics Data System (ADS)

    Malov, Alexander N.; Neupokoeva, Anna V.; Morozov, Alexey N.; Timoshenko, Elena A.

    2016-11-01

    A craquelure structure of protein film as indicator of macromolecule state is discussing. Craquelure is a network of fine cracks or crackles on the surface of a painting, caused chiefly by shrinkage of paint film or varnish. The actions of laser radiation in the red and green spectral region on the protein film craquelure structure by the example of albumin are considering. It is experimentally shown that after drying the protein layer a craquelure pattern (variety of cracks in the layer) is formed with the parameters strongly modified by the laser action and depending on the time (energy density) of exposure. The threshold energy of laser action is defined; it does not depend on wavelength significantly.