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Sample records for chaperone-effector protein complex

  1. Detecting overlapping protein complexes in protein-protein interaction networks

    OpenAIRE

    Nepusz, Tamás; Yu, Haiyuan; Paccanaro, Alberto

    2012-01-01

    We introduce clustering with overlapping neighborhood expansion (ClusterONE), a method for detecting potentially overlapping protein complexes from protein-protein interaction data. ClusterONE-derived complexes for several yeast data sets showed better correspondence with reference complexes in the Munich Information Center for Protein Sequence (MIPS) catalog and complexes derived from the Saccharomyces Genome Database (SGD) than the results of seven popular methods. The results also showed a...

  2. Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

    OpenAIRE

    Peng Liu; Lei Yang; Daming Shi; Xianglong Tang

    2015-01-01

    A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction net...

  3. The fifth adaptor protein complex.

    OpenAIRE

    Jennifer Hirst; Barlow, Lael D.; Gabriel Casey Francisco; Sahlender, Daniela A.; Seaman, Matthew N.J.; Dacks, Joel B.; Robinson, Margaret S.

    2011-01-01

    Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent manno...

  4. The Fifth Adaptor Protein Complex

    OpenAIRE

    Hirst, Jennifer; D. Barlow, Lael; Francisco, Gabriel Casey; Sahlender, Daniela A.; Seaman, Matthew N.J.; Dacks, Joel B.; Robinson, Margaret S.

    2011-01-01

    Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent manno...

  5. The fifth adaptor protein complex.

    Directory of Open Access Journals (Sweden)

    Jennifer Hirst

    2011-10-01

    Full Text Available Adaptor protein (AP complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.

  6. Adaptor protein complexes and intracellular transport

    OpenAIRE

    2014-01-01

    The AP (adaptor protein) complexes are heterotetrameric protein complexes that mediate intracellular membrane trafficking along endocytic and secretory transport pathways. There are five different AP complexes: AP-1, AP-2 and AP-3 are clathrin-associated complexes; whereas AP-4 and AP-5 are not. These five AP complexes localize to different intracellular compartments and mediate membrane trafficking in distinct pathways. They recognize and concentrate cargo proteins into vesicular carriers th...

  7. Ontology integration to identify protein complex in protein interaction networks

    OpenAIRE

    Yang Zhihao; Lin Hongfei; Xu Bo

    2011-01-01

    Abstract Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity metho...

  8. Protein-protein complexation in bioluminescence

    OpenAIRE

    Titushin, Maxim S.; Feng, Yingang; Lee, John; Vysotski, Eugene S.; Liu, Zhi-jie

    2011-01-01

    In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an “accessory protein” whereby a stored substrate is efficiently delivered to the bioluminescent enzyme lucife...

  9. Ontology integration to identify protein complex in protein interaction networks

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    Yang Zhihao

    2011-10-01

    Full Text Available Abstract Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches.

  10. 3D complex: a structural classification of protein complexes.

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    Emmanuel D Levy

    2006-11-01

    Full Text Available Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

  11. Protein Complex Purification by Affinity Capture.

    Science.gov (United States)

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  12. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes

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    Heike Angerer

    2015-02-01

    Full Text Available In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine motif proteins (LYRMs of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6 or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1 of the oxidative phosphorylation (OXPHOS core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria.

  13. Widespread cotranslational formation of protein complexes.

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    Caia D S Duncan

    2011-12-01

    Full Text Available Most cellular processes are conducted by multi-protein complexes. However, little is known about how these complexes are assembled. In particular, it is not known if they are formed while one or more members of the complexes are being translated (cotranslational assembly. We took a genomic approach to address this question, by systematically identifying mRNAs associated with specific proteins. In a sample of 31 proteins from Schizosaccharomyces pombe that did not contain RNA-binding domains, we found that ∼38% copurify with mRNAs that encode interacting proteins. For example, the cyclin-dependent kinase Cdc2p associates with the rum1 and cdc18 mRNAs, which encode, respectively, an inhibitor of Cdc2p kinase activity and an essential regulator of DNA replication. Both proteins interact with Cdc2p and are key cell cycle regulators. We obtained analogous results with proteins with different structures and cellular functions (kinesins, protein kinases, transcription factors, proteasome components, etc.. We showed that copurification of a bait protein and of specific mRNAs was dependent on the presence of the proteins encoded by the interacting mRNAs and on polysomal integrity. These results indicate that these observed associations reflect the cotranslational interaction between the bait and the nascent proteins encoded by the interacting mRNAs. Therefore, we show that the cotranslational formation of protein-protein interactions is a widespread phenomenon.

  14. The Claudin Megatrachea Protein Complex*

    Science.gov (United States)

    Jaspers, Martin H. J.; Nolde, Kai; Behr, Matthias; Joo, Seol-hee; Plessmann, Uwe; Nikolov, Miroslav; Urlaub, Henning; Schuh, Reinhard

    2012-01-01

    Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates. PMID:22930751

  15. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    OpenAIRE

    Selvaraj S; Jayaram B; Saranya N; Gromiha M; Fukui Kazuhiko

    2011-01-01

    Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such...

  16. Complex coacervation of supercharged proteins with polyelectrolytes.

    Science.gov (United States)

    Obermeyer, Allie C; Mills, Carolyn E; Dong, Xue-Hui; Flores, Romeo J; Olsen, Bradley D

    2016-04-21

    Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.

  17. Analysis of correlations between protein complex and protein-protein interaction and mRNA expression

    Institute of Scientific and Technical Information of China (English)

    CAI Lun; XUE Hong; LU Hongchao; ZHAO Yi; ZHU Xiaopeng; BU Dongbo; LING Lunjiang; CHEN Runsheng

    2003-01-01

    Protein-protein interaction is a physical interaction of two proteins in living cells. In budding yeast Saccharomyces cerevisiae, large-scale protein-protein interaction data have been obtained through high-throughput yeast two-hybrid systems (Y2H) and protein complex purification techniques based on mass-spectrometry. Here, we collect 11855 interactions between total 2617 proteins. Through seriate genome-wide mRNA expression data, similarity between two genes could be measured. Protein complex data can also be obtained publicly and can be translated to pair relationship that any two proteins can only exist in the same complex or not. Analysis of protein complex data, protein-protein interaction data and mRNA expression data can elucidate correlations between them. The results show that proteins that have interactions or similar expression patterns have a higher possibility to be in the same protein complex than randomized selected proteins, and proteins which have interactions and similar expression patterns are even more possible to exist in the same protein complex. The work indicates that comprehensive integration and analysis of public large-scale bioinformatical data, such as protein complex data, protein-protein interaction data and mRNA expression data, may help to uncover their relationships and common biological information underlying these data. The strategies described here may help to integrate and analyze other functional genomic and proteomic data, such as gene expression profiling, protein-localization mapping and large-scale phenotypic data, both in yeast and in other organisms.

  18. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    Science.gov (United States)

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  19. High throughput protein-protein interaction data: clues for the architecture of protein complexes

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    Pang Chi

    2008-11-01

    Full Text Available Abstract Background High-throughput techniques are becoming widely used to study protein-protein interactions and protein complexes on a proteome-wide scale. Here we have explored the potential of these techniques to accurately determine the constituent proteins of complexes and their architecture within the complex. Results Two-dimensional representations of the 19S and 20S proteasome, mediator, and SAGA complexes were generated and overlaid with high quality pairwise interaction data, core-module-attachment classifications from affinity purifications of complexes and predicted domain-domain interactions. Pairwise interaction data could accurately determine the members of each complex, but was unexpectedly poor at deciphering the topology of proteins in complexes. Core and module data from affinity purification studies were less useful for accurately defining the member proteins of these complexes. However, these data gave strong information on the spatial proximity of many proteins. Predicted domain-domain interactions provided some insight into the topology of proteins within complexes, but was affected by a lack of available structural data for the co-activator complexes and the presence of shared domains in paralogous proteins. Conclusion The constituent proteins of complexes are likely to be determined with accuracy by combining data from high-throughput techniques. The topology of some proteins in the complexes will be able to be clearly inferred. We finally suggest strategies that can be employed to use high throughput interaction data to define the membership and understand the architecture of proteins in novel complexes.

  20. CPL:Detecting Protein Complexes by Propagating Labels on Protein-Protein Interaction Network

    Institute of Scientific and Technical Information of China (English)

    代启国; 郭茂祖; 刘晓燕; 滕志霞; 王春宇

    2014-01-01

    Proteins usually bind together to form complexes, which play an important role in cellular activities. Many graph clustering methods have been proposed to identify protein complexes by finding dense regions in protein-protein interaction networks. We present a novel framework (CPL) that detects protein complexes by propagating labels through interactions in a network, in which labels denote complex identifiers. With proper propagation in CPL, proteins in the same complex will be assigned with the same labels. CPL does not make any strong assumptions about the topological structures of the complexes, as in previous methods. The CPL algorithm is tested on several publicly available yeast protein-protein interaction networks and compared with several state-of-the-art methods. The results suggest that CPL performs better than the existing methods. An analysis of the functional homogeneity based on a gene ontology analysis shows that the detected complexes of CPL are highly biologically relevant.

  1. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Directory of Open Access Journals (Sweden)

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  2. An Algorithm for Finding Functional Modules and Protein Complexes in Protein-Protein Interaction Networks

    OpenAIRE

    Guangyu Cui; Yu Chen; De-Shuang Huang; Kyungsook Han

    2008-01-01

    Biological processes are often performed by a group of proteins rather than by individual proteins, and proteins in a same biological group form a densely connected subgraph in a protein-protein interaction network. Therefore, finding a densely connected subgraph provides useful information to predict the function or protein complex of uncharacterized proteins in the highly connected subgraph. We have developed an efficient algorithm and program for finding cliques and near-cliques in a prote...

  3. Isolation of proteins and protein complexes by immunoprecipitation.

    Science.gov (United States)

    Kaboord, Barbara; Perr, Maria

    2008-01-01

    Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity method. The traditional method of incubating the IP antibody with the sample and sequentially binding to Protein A or G agarose beads (resin) facilitates the most efficient target antigen recovery. However, this approach results in the target protein becoming contaminated with the IP antibody that can interfere with downstream analyses. The orientated affinity method uses Protein A or G beads to serve as an anchor to which the IP antibody is crosslinked thereby preventing the antibody from co-eluting with the target protein. Similarly, the direct affinity method also immobilizes the IP antibody except in this case it is directly attached to a chemically activated support. Both methods prevent co-elution of the IP antibody enabling reuse of the immunomatrix. All three approaches have unique advantages and can also be used for co-immunoprecipitation to study protein:protein interactions and investigate the functional proteome.

  4. Characterization of Protein Complexes and Subcomplexes in Protein-Protein Interaction Databases

    OpenAIRE

    Nazar Zaki; Elfadil A. Mohamed; Antonio Mora

    2015-01-01

    The identification and characterization of protein complexes implicated in protein-protein interaction data are crucial to the understanding of the molecular events under normal and abnormal physiological conditions. This paper provides a novel characterization of subcomplexes in protein interaction databases, stressing definition and representation issues, quantification, biological validation, network metrics, motifs, modularity, and gene ontology (GO) terms. The paper introduces the concep...

  5. HKC: An Algorithm to Predict Protein Complexes in Protein-Protein Interaction Networks

    OpenAIRE

    Xiaomin Wang; Zhengzhi Wang; Jun Ye

    2011-01-01

    With the availability of more and more genome-scale protein-protein interaction (PPI) networks, research interests gradually shift to Systematic Analysis on these large data sets. A key topic is to predict protein complexes in PPI networks by identifying clusters that are densely connected within themselves but sparsely connected with the rest of the network. In this paper, we present a new topology-based algorithm, HKC, to detect protein complexes in genome-scale PPI networks. HKC mainly use...

  6. The evolution of protein complexes by duplication of homomeric interactions

    OpenAIRE

    Pereira Leal, J.B.; Levy, E.D.; van de Kamp, C.; Teichmann, S.A.

    2007-01-01

    BACKGROUND: Cellular functions are accomplished by the concerted actions of functional modules. The mechanisms driving the emergence and evolution of these modules are still unclear. Here we investigate the evolutionary origins of protein complexes, modules in physical protein-protein interaction networks. RESULTS: We studied protein complexes in Saccharomyces cerevisiae, complexes of known three-dimensional structure in the Protein Data Bank and clusters of pairwise protein interactions in t...

  7. Evolution of protein complexes by duplication of homomeric interactions

    OpenAIRE

    Pereira-Leal, Jose B; Levy, Emmanuel D; Kamp, Christel; Teichmann, Sarah A.

    2007-01-01

    Background Cellular functions are accomplished by the concerted actions of functional modules. The mechanisms driving the emergence and evolution of these modules are still unclear. Here we investigate the evolutionary origins of protein complexes, modules in physical protein-protein interaction networks. Results We studied protein complexes in Saccharomyces cerevisiae, complexes of known three-dimensional structure in the Protein Data Bank and clusters of pairwise protein interactions in the...

  8. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking.

    Science.gov (United States)

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  9. Targeted Interactomics in Plants Through Protein Complex Isolation

    Institute of Scientific and Technical Information of China (English)

    Geert De Jaeger

    2012-01-01

    TAPtag technology is the most widely applied tool to pick up in situ protein interactions in a proteome wide setting.Our research team has developed a versatile TAP technology platform for protein complex isolation from plants.We isolated complexes for hundreds of proteins and extensively demonstrated the power of our technology for protein discovery,functional analysis of proteins and protein complexes,and the modelling of protein networks.Complexes are purified from Arabidopsis cell suspension cultures or seedlings and we are currently translating the technology towards crop plants to bring complex purification in a developmental context.Besides protein complexes,we are deriving protocol variations to isolate chromatin complexes.

  10. How proteins get in touch: Interface prediction and docking of protein complexes

    NARCIS (Netherlands)

    de Vries, S.J.

    2009-01-01

    Proteins are the wheels and mill stones of the complex machinery that underlies human life. In carrying out their functions, proteins work in close association with other proteins, forming protein complexes. A huge network of protein-protein interactions enables the cell to respond quickly to change

  11. Discover Protein Complexes in Protein-Protein Interaction Networks Using Parametric Local Modularity

    OpenAIRE

    Tan Kai; Kim Jongkwang

    2010-01-01

    Abstract Background Recent advances in proteomic technologies have enabled us to create detailed protein-protein interaction maps in multiple species and in both normal and diseased cells. As the size of the interaction dataset increases, powerful computational methods are required in order to effectively distil network models from large-scale interactome data. Results We present an algorithm, miPALM (Module Inference by Parametric Local Modularity), to infer protein complexes in a protein-pr...

  12. Engineering of complex protein sialylation in plants

    Science.gov (United States)

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-01-01

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  13. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Hao Wu

    Full Text Available In this paper, we present a novel rough-fuzzy clustering (RFC method to detect overlapping protein complexes in protein-protein interaction (PPI networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks.

  14. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    Science.gov (United States)

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks. PMID:24642838

  15. Analysis of endogenous protein complexes by mass spectrometry

    NARCIS (Netherlands)

    Synowsky, S.A.

    2008-01-01

    Proteins are organized in large protein complexes that form an extensive network in the cell. They are the most versatile macromolecule in the cell and the interactions between each other are highly directed and essential for most cellular functions. The activity of protein complexes is in turn freq

  16. Inferring protein inverted question markprotein interaction complexes from immunoprecipitation data

    NARCIS (Netherlands)

    J. Kutzera; H.C.J. Hoefsloot; A. Malovannaya; A.B. Smit; I. van Mechelen; A.K. Smilde

    2013-01-01

    BACKGROUND: Protein inverted question markprotein interactions in cells are widely explored using small inverted question markscale experiments. However, the search for protein complexes and their interactions in data from high throughput experiments such as immunoprecipitation is still a challenge.

  17. Accumulation of small protein molecules in a macroscopic complex coacervate

    NARCIS (Netherlands)

    Lindhoud, S.; Claessens, M.M.A.E.

    2016-01-01

    To obtain insight into the accumulation of proteins into macroscopic complex coacervate phases, the lysozyme concentration in complex coacervates containing the cationic polyelectrolyte poly-(N,N dimethylaminoethyl methacrylate) and the anionic polyelectrolyte polyacrylic acid was investigated as a

  18. Affinity Purification of Protein Complexes Using TAP Tags

    Science.gov (United States)

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  19. Recording information on protein complexes in an information management system.

    Science.gov (United States)

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  20. Principles of assembly reveal a periodic table of protein complexes.

    Science.gov (United States)

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  1. Recording information on protein complexes in an information management system.

    Science.gov (United States)

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.

  2. Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry*

    OpenAIRE

    Kaake, Robyn M; Wang, Xiaorong; Huang, Lan

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand ho...

  3. Studies of protein-protein and protein-RNA complexes by mass spectrometry

    International Nuclear Information System (INIS)

    Electrospray ionization mass spectrometry (ESI-MS) enables the determination of not only the molecular sizes of non-covalent macromolecular complexes, but also the binding affinities. This paper shows the advantages of ESI-MS over other techniques in structural studies of biological macromolecular complexes, using as an example our recent analysis of TRAP, trp RNA-binding attenuation protein, and its complexes with RNA and protein ligands. TRAP and its regulator anti-TRAP protein (AT) play the principal roles in controlling tryptophan synthesis in Bacillus species. We have characterized both wild-type (wt) and mutant TRAP from B. stearothermophilus, and their complexes with RNA or AT by ESI-MS. Wild-type TRAP forms homo-11mer rings. The mutant used carries three copies of the TRAP monomer on a single polypeptide chain, so that it associates to form a 12mer ring with four polypeptides. Mass spectra showed that both the wt TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA. The crystal structure of wt TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, ESI-MS of wild-type TRAP mixed with AT shows four species with different binding stoichiometries, and the complex observed by crystallography represents only a minor species in solution; most of the TRAP remains in an 11mer ring form. Mass spectra of mutant TRAP showed only a single species, TRAP 12mer + six copies of AT trimer, which is observed by crystallography. The crystal structure of the TRAP-AT complexes shows that only a 12mer TRAP ring can bind with six copies of AT trimer. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, while ESI-MS can take a 'snapshot' of all the species in solution. (author)

  4. Alpha complexes in protein structure prediction

    DEFF Research Database (Denmark)

    Winter, Pawel; Fonseca, Rasmus

    2015-01-01

    Reducing the computational effort and increasing the accuracy of potential energy functions is of utmost importance in modeling biological systems, for instance in protein structure prediction, docking or design. Evaluating interactions between nonbonded atoms is the bottleneck of such computatio...

  5. Protein Packing Quality Using Delaunay Complexes

    DEFF Research Database (Denmark)

    Fonseca, Rasmus; Winter, Pawel; Karplus, Kevin

    2011-01-01

    A new method for estimating the packing quality of protein structures is presented. Atoms in high quality protein crystal structures are very uniformly distributed which is difficult to reproduce using structure prediction methods. Packing quality measures can therefore be used to assess structur...... respectively. Our results show that the developed method is correlated to the well-established RosettaHoles2 but three times faster....

  6. Unwinding Protein Complexes in ALTernative Telomere Maintenance

    OpenAIRE

    Bhattacharyya, Saumitri; Sandy, April; Groden, Joanna

    2010-01-01

    Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA-binding proteins and a number of three dimensional nucleic acid structures including G-quartets and D-loops. A number of studies suggest that the BLM and WRN recQ-like helicases play important roles in recombination-mediated mechanisms of telomere elongation or Alternative Lengthening of Telomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and ...

  7. Recognition of protein complexation based on hydrophobicity distribution

    OpenAIRE

    Banach, Mateusz; Roterman, Irena

    2009-01-01

    The identification of the surface area able to generate the protein-protein complexation ligand and ion ligation is critical for the recognition of the biological function of particular proteins. The technique based on the analysis of the irregularity of hydrophobicity distribution is used as the criterion for the recognition of the interaction regions. Particularly, the exposure of hydrophobic residues on the surface of protein as well as the localization of the hydrophilic residues in the h...

  8. Recording information on protein complexes in an information management system

    Science.gov (United States)

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  9. Protein Connectivity and Protein Complexity Promotes Human Gene Duplicability in a Mutually Exclusive Manner

    OpenAIRE

    Bhattacharya, Tanusree; Ghosh, Tapash Chandra

    2010-01-01

    It has previously been reported that protein complexity (i.e. number of subunits in a protein complex) is negatively correlated to gene duplicability in yeast as well as in humans. However, unlike in yeast, protein connectivity in a protein–protein interaction network has a positive correlation with gene duplicability in human genes. In the present study, we have analyzed 1732 human and 1269 yeast proteins that are present both in a protein–protein interaction network as well as in a protein ...

  10. Quantifying the energetics of cooperativity in a ternary protein complex

    DEFF Research Database (Denmark)

    Andersen, Peter S; Schuck, Peter; Sundberg, Eric J;

    2002-01-01

    binary reactions that make up the overall complex. This is due, in large part, to cooperative interactions between separate protein domains. Thus, a full understanding of multiprotein complexes requires the quantitative analysis of cooperativity. We have used surface plasmon resonance techniques...... and mathematical modeling to describe the energetics of cooperativity in a trimolecular protein complex. As a model system for quantifying cooperativity, we studied the ternary complex formed by the simultaneous interaction of a superantigen with major histocompatibility complex and T cell receptor, for which...... a structural model is available. This system exhibits positive and negative cooperativity, as well as augmentation of the temperature dependence of binding kinetics upon the cooperative interaction of individual protein components in the complex. Our experimental and theoretical analysis may be applicable...

  11. Carbohydrate – protein complex of the waste of climacoptera obtusifolia

    Directory of Open Access Journals (Sweden)

    G. Seitimova

    2013-05-01

    Full Text Available Extract from Climacoptera obtusifolia family Chenopodiaceae has antidiabetic activity. For the first time carbohydrate-protein complex of the waste from Climacoptera obtusifolia was studied. It was found that the quantity of extractive substances with 80% ethanol in aerial part – 52;6% and in the waste – 12;35%. The technique of separation of the carbohydrate-protein complex from the waste from Climacoptera obtusifolia is developed by means of classical and physical-chemical methods. The composition of carbohydrate-protein complex was identified: oligosaccharide; polysaccharide and two glycoproteins.

  12. Protein-DNA complexes: specificity and DNA readout mechanisms

    Directory of Open Access Journals (Sweden)

    Shestopalova A. V.

    2011-02-01

    Full Text Available Protein-nucleic acid recognition is essential in a number of cellular processes, in particular, gene regulation, DNA replication and compaction. Studies on the recognition mechanisms show that DNA sequence carries information which is read out by proteins that selectively bind to specific DNA sites. The review is focused on the processes taking place during formation of specific and nonspecific complexes of proteins and DNA. Special attention is paid to direct and indirect mechanisms of sequence-specific recognition. Several examples of protein-nucleic acid complexes are given to illustrate the variety of recognition mechanisms

  13. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

    OpenAIRE

    Eileen Marie Hanna; Nazar Zaki; Amr Amin

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, ...

  14. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

    OpenAIRE

    Putty-Reddy Sudhir; Chung-Hsuan Chen

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. T...

  15. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    Energy Technology Data Exchange (ETDEWEB)

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  16. Protein Connectivity in Chemotaxis Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Stephan Eismann

    2015-12-01

    Full Text Available The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.

  17. Complex formation by the Drosophila MSL proteins: role of the MSL2 RING finger in protein complex assembly.

    OpenAIRE

    Copps, K.; Richman, R.; Lyman, L M; Chang, K A; Rampersad-Ammons, J; Kuroda, M I

    1998-01-01

    Drosophila MSL proteins are thought to act within a complex to elevate transcription from the male X chromosome. We found that the MSL1, MSL2 and MSL3 proteins are associated in immunoprecipitations, chromatographic steps and in the yeast two-hybrid system, but that the MLE protein is not tightly complexed in these assays. We focused our analysis on the MSL2-MSL1 interaction, which is postulated to play a critical role in MSL complex association with the X chromosome. Using a modified two-hyb...

  18. Structural study of coacervation in protein-polyelectrolyte complexes

    Science.gov (United States)

    Chodankar, S.; Aswal, V. K.; Kohlbrecher, J.; Vavrin, R.; Wagh, A. G.

    2008-09-01

    Coacervation is a dense liquid-liquid phase separation and herein we report coacervation of protein bovine serum albumin (BSA) in the presence of polyelectrolyte sodium polystyrene sulfonate (NaPSS) under varying solution conditions. Small-angle neutron scattering (SANS) measurements have been performed on above protein-polyelectrolyte complexes to study the structural evolution of the process that leads to coacervation and the phase separated coacervate as a function of solution pH , protein-polyelectrolyte ratio and ionic strength. SANS study prior to phase separation on the BSA-NaPSS complex shows a fractal structure representing a necklace model of protein macromolecules randomly distributed along the polystyrene sulfonate chain. The fractal dimension of the complex decreases as pH is shifted away from the isoelectric point (˜4.7) of BSA protein, which indicates the decrease in the compactness of the complex structure due to increase in the charge repulsion between the protein macromolecules bound to the polyelectrolyte. Concentration-dependence studies of the polyelectrolyte in the complex suggest coexistence of two populations of polyelectrolytes, first one fully saturated with proteins and another one free from proteins. Coacervation phase has been obtained through the turbidity measurement by varying pH of the aqueous solution containing protein and polyelectrolyte from neutral to acidic regime to get them to where the two components are oppositely charged. The spontaneous formation of coacervates is observed for pH values less than 4. SANS study on coacervates shows two length scales related to complex aggregations (mesh size and overall extent of the complex) hierarchically branched to form a larger network. The mesh size represents the distance between cross-linked points in the primary complex, which decreases with increase in ionic strength and remains the same on varying the protein-polyelectrolyte ratio. On the other hand, the overall extent of the

  19. Blotting protein complexes from native gels to electron microscopy grids.

    Science.gov (United States)

    Knispel, Roland Wilhelm; Kofler, Christine; Boicu, Marius; Baumeister, Wolfgang; Nickell, Stephan

    2012-01-08

    We report a simple and generic method for the direct transfer of protein complexes separated by native gel electrophoresis to electron microscopy grids. After transfer, sufficient material remains in the gel for identification and characterization by mass spectrometry. The method should facilitate higher-throughput single-particle analysis by substantially reducing the time needed for protein purification, as demonstrated for three complexes from Thermoplasma acidophilum.

  20. Molecular simulations of carbohydrate-protein complexes

    OpenAIRE

    Eid, Sameh Mansour Abbas

    2013-01-01

    I. Generation and validation of a free-energy model for carbohydrate binding. Carbohy-drates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the stru-cture-based design of carbohydrate-based ligands. To this end, we assembled a diverse data set of 316 carbohydrate–protein crystal structu...

  1. Optimized Affinity Capture of Yeast Protein Complexes.

    Science.gov (United States)

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596

  2. Traveling-wave ion mobility mass spectrometry of protein complexes

    DEFF Research Database (Denmark)

    Salbo, Rune; Bush, Matthew F; Naver, Helle;

    2012-01-01

    The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of...

  3. Accuracy improvement in protein complex prediction from protein interaction networks by refining cluster overlaps

    OpenAIRE

    Chiam Tak; Cho Young-Rae

    2012-01-01

    Abstract Background Recent computational techniques have facilitated analyzing genome-wide protein-protein interaction data for several model organisms. Various graph-clustering algorithms have been applied to protein interaction networks on the genomic scale for predicting the entire set of potential protein complexes. In particular, the density-based clustering algorithms which are able to generate overlapping clusters, i.e. the clusters sharing a set of nodes, are well-suited to protein co...

  4. Complex protein nanopatterns over large areas via colloidal lithography

    DEFF Research Database (Denmark)

    Kristensen, Stine H; Pedersen, Gitte Albinus; Ogaki, Ryosuke;

    2013-01-01

    matrix proteins (vitronectin) or cellular ligands (the extracellular domain of E-cadherin) in the nanopatterns, whereas the selective poly(l-lysine)–poly(ethylene glycol) functionalization of the SiO2 matrix renders it protein repellent. Cell studies, as a proof of principle, demonstrate the potential...... are used to generate complex protein nanopatterns over large areas. Simple circular patches or more complex ring structures are produced in addition to hierarchical patterns of smaller patches. The gold regions are modified through alkanethiol chemistry, which enables the preparation of extracellular...

  5. Protein Complex Production from the Drug Discovery Standpoint.

    Science.gov (United States)

    Moarefi, Ismail

    2016-01-01

    Small molecule drug discovery critically depends on the availability of meaningful in vitro assays to guide medicinal chemistry programs that are aimed at optimizing drug potency and selectivity. As it becomes increasingly evident, most disease relevant drug targets do not act as a single protein. In the body, they are instead generally found in complex with protein cofactors that are highly relevant for their correct function and regulation. This review highlights selected examples of the increasing trend to use biologically relevant protein complexes for rational drug discovery to reduce costly late phase attritions due to lack of efficacy or toxicity.

  6. Complexation between dodecyl sulfate surfactant and zein protein in solution.

    Science.gov (United States)

    Ruso, Juan M; Deo, Namita; Somasundaran, P

    2004-10-12

    Interactions between sodium dodecyl sulfate and zein protein, a model system for the understanding of the effect of surfactants on skin, were investigated using a range of techniques involving UV-vis spectroscopy, TOC (total organic carbon analysis), electrophoresis, and static and dynamic light scattering. Zein protein was solubilized by SDS. The adsorption of SDS onto insoluble protein fraction caused the zeta potential of the complex to become more negative. From these values, we calculated the Gibbs energy of absorption, which decreases when the SDS concentration is raised. Finally the structure of the complex, based on the analysis by static and dynamic light scattering, is proposed to be rod like.

  7. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes.

    Directory of Open Access Journals (Sweden)

    Jiawei Luo

    Full Text Available Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins.In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC, based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID, of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification.Experimental results based on three different PPI(protein-protein interaction networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC.LIDC is more effective for the prediction of essential proteins than other recently developed methods.

  8. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

    OpenAIRE

    Jun Ren; Wei Zhou; Jianxin Wang

    2014-01-01

    Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clusterin...

  9. A holistic molecular docking approach for predicting protein-protein complex structure

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A holistic protein-protein molecular docking approach,HoDock,was established,composed of such steps as binding site prediction,initial complex structure sampling,refined complex structure sampling,structure clustering,scoring and final structure selection.This article explains the detailed steps and applications for CAPRI Target 39.The CAPRI result showed that three predicted binding site residues,A191HIS,B512ARG and B531ARG,were correct,and there were five submitted structures with a high fraction of correct receptor-ligand interface residues,indicating that this docking approach may improve prediction accuracy for protein-protein complex structures.

  10. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters.

    Science.gov (United States)

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present "DyCluster", a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  11. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters.

    Directory of Open Access Journals (Sweden)

    Eileen Marie Hanna

    Full Text Available Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present "DyCluster", a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster.

  12. Identifying hierarchical and overlapping protein complexes based on essential protein-protein interactions and "seed-expanding" method.

    Science.gov (United States)

    Ren, Jun; Zhou, Wei; Wang, Jianxin

    2014-01-01

    Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and "seed-expanding." First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes. PMID:25143945

  13. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

    Directory of Open Access Journals (Sweden)

    Jun Ren

    2014-01-01

    Full Text Available Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes.

  14. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

    Science.gov (United States)

    Ren, Jun; Zhou, Wei; Wang, Jianxin

    2014-01-01

    Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes. PMID:25143945

  15. Emergence of Complexity in Protein Functions and Metabolic Networks

    Science.gov (United States)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  16. Decomposition of overlapping protein complexes: A graph theoretical method for analyzing static and dynamic protein associations

    Directory of Open Access Journals (Sweden)

    Guimarães Katia S

    2006-04-01

    Full Text Available Abstract Background Most cellular processes are carried out by multi-protein complexes, groups of proteins that bind together to perform a specific task. Some proteins form stable complexes, while other proteins form transient associations and are part of several complexes at different stages of a cellular process. A better understanding of this higher-order organization of proteins into overlapping complexes is an important step towards unveiling functional and evolutionary mechanisms behind biological networks. Results We propose a new method for identifying and representing overlapping protein complexes (or larger units called functional groups within a protein interaction network. We develop a graph-theoretical framework that enables automatic construction of such representation. We illustrate the effectiveness of our method by applying it to TNFα/NF-κB and pheromone signaling pathways. Conclusion The proposed representation helps in understanding the transitions between functional groups and allows for tracking a protein's path through a cascade of functional groups. Therefore, depending on the nature of the network, our representation is capable of elucidating temporal relations between functional groups. Our results show that the proposed method opens a new avenue for the analysis of protein interaction networks.

  17. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    Science.gov (United States)

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  18. Displacement affinity chromatography of protein phosphatase one (PP1 complexes

    Directory of Open Access Journals (Sweden)

    Gourlay Robert

    2008-11-01

    Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

  19. Economy of operon formation: cotranscription minimizes shortfall in protein complexes.

    Science.gov (United States)

    Sneppen, Kim; Pedersen, Steen; Krishna, Sandeep; Dodd, Ian; Semsey, Szabolcs

    2010-01-01

    Genes of prokaryotes and Archaea are often organized in cotranscribed groups, or operons. In contrast, eukaryotic genes are generally transcribed independently. Here we show that there is a substantial economic gain for the cell to cotranscribe genes encoding protein complexes because it synchronizes the fluctuations, or noise, in the levels of the different components. This correlation substantially reduces the shortfall in production of the complex. This benefit is relatively large in small cells such as bacterial cells, in which there are few mRNAs and proteins per cell, and is diminished in larger cells such as eukaryotic cells. PMID:20877578

  20. Biclustering Protein Complex Interactions with a Biclique FindingAlgorithm

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Chris; Zhang, Anne Ya; Holbrook, Stephen

    2006-12-01

    Biclustering has many applications in text mining, web clickstream mining, and bioinformatics. When data entries are binary, the tightest biclusters become bicliques. We propose a flexible and highly efficient algorithm to compute bicliques. We first generalize the Motzkin-Straus formalism for computing the maximal clique from L{sub 1} constraint to L{sub p} constraint, which enables us to provide a generalized Motzkin-Straus formalism for computing maximal-edge bicliques. By adjusting parameters, the algorithm can favor biclusters with more rows less columns, or vice verse, thus increasing the flexibility of the targeted biclusters. We then propose an algorithm to solve the generalized Motzkin-Straus optimization problem. The algorithm is provably convergent and has a computational complexity of O(|E|) where |E| is the number of edges. It relies on a matrix vector multiplication and runs efficiently on most current computer architectures. Using this algorithm, we bicluster the yeast protein complex interaction network. We find that biclustering protein complexes at the protein level does not clearly reflect the functional linkage among protein complexes in many cases, while biclustering at protein domain level can reveal many underlying linkages. We show several new biologically significant results.

  1. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    Science.gov (United States)

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  2. Unwrapping of DNA-protein complexes under external stretching

    OpenAIRE

    Sakaue, Takahiro; Löwen, Hartmut

    2004-01-01

    A DNA-protein complex modelled by a semiflexible chain and an attractive spherical core is studied in the situation when an external stretching force is acting on one end monomer of the chain while the other end monomer is kept fixed in space. Without stretching force, the chain is wrapped around the core. By applying an external stretching force, unwrapping of the complex is induced. We study the statics and the dynamics of the unwrapping process by computer simulation and simple phenomenolo...

  3. Information-driven modeling of protein-peptide complexes.

    Science.gov (United States)

    Trellet, Mikael; Melquiond, Adrien S J; Bonvin, Alexandre M J J

    2015-01-01

    Despite their biological importance in many regulatory processes, protein-peptide recognition mechanisms are difficult to study experimentally at the structural level because of the inherent flexibility of peptides and the often transient interactions on which they rely. Complementary methods like biomolecular docking are therefore required. The prediction of the three-dimensional structure of protein-peptide complexes raises unique challenges for computational algorithms, as exemplified by the recent introduction of protein-peptide targets in the blind international experiment CAPRI (Critical Assessment of PRedicted Interactions). Conventional protein-protein docking approaches are often struggling with the high flexibility of peptides whose short sizes impede protocols and scoring functions developed for larger interfaces. On the other side, protein-small ligand docking methods are unable to cope with the larger number of degrees of freedom in peptides compared to small molecules and the typically reduced available information to define the binding site. In this chapter, we describe a protocol to model protein-peptide complexes using the HADDOCK web server, working through a test case to illustrate every steps. The flexibility challenge that peptides represent is dealt with by combining elements of conformational selection and induced fit molecular recognition theories. PMID:25555727

  4. Effects of ionizing radiations on DNA-protein complexes

    International Nuclear Information System (INIS)

    The radio-induced destruction of DNA-protein complexes may have serious consequences for systems implicated in important cellular functions. The first system which has been studied is the lactose operon system, that regulates gene expression in Escherichia coli. First of all, the repressor-operator complex is destroyed after irradiation of the complex or of the protein alone. The damaging of the domain of repressor binding to DNA (headpiece) has been demonstrated and studied from the point of view of peptide chain integrity, conformation and amino acids damages. Secondly, dysfunctions of the in vitro induction of an irradiated repressor-unirradiated DNA complex have been observed. These perturbations, due to a decrease of the number of inducer binding sites, are correlated to the damaging of tryptophan residues. Moreover, the inducer protects the repressor when they are irradiated together, both by acting as a scavenger in the bulk, and by the masking of its binding site on the protein. The second studied system is formed by Fpg (for Formamido pyrimidine glycosylase), a DNA repair protein and a DNA with an oxidative lesion. The results show that irradiation disturbs the repair both by decreasing its efficiency of DNA lesion recognition and binding, and by altering its enzymatic activity. (author)

  5. Evolutionary Optimization of Kernel Weights Improves Protein Complex Comembership Prediction

    NARCIS (Netherlands)

    Hulsman, M.; Reinders, M.J.T.; De Ridder, D.

    2008-01-01

    In recent years, more and more high-throughput data sources useful for protein complex prediction have become available (e.g., gene sequence, mRNA expression, and interactions). The integration of these different data sources can be challenging. Recently, it has been recognized that kernel-based cla

  6. Encapsulation of Protein-Polysaccharide HIP Complex in Polymeric Nanoparticles

    Directory of Open Access Journals (Sweden)

    Ripal Gaudana

    2011-01-01

    Full Text Available The objective of the present study is to formulate and characterize a nanoparticulate-based formulation of a macromolecule in a hydrophobic ion pairing (HIP complex form. So far, HIP complexation approach has been studied only for proteins with molecular weight of 10–20 kDa. Hence, we have selected bovine serum albumin (BSA having higher molecular weight (66.3 kDa as a model protein and dextran sulphate (DS as a complexing polymer to generate HIP complex. We have prepared and optimized the HIP complex formation process of BSA with DS. Ionic interactions between basic amino acids of BSA with sulphate groups of DS were confirmed by FTIR analysis. Further, nanoparticles were prepared and characterized with respect to size and surface morphology. We observed significant entrapment of BSA in nanoparticles prepared with minimal amounts of PLGA polymer. Finally, results of circular dichroism and intrinsic fluorescence assay have clearly indicated that HIP complexation and method of nanoparticle preparation did not alter the secondary and tertiary structures of BSA.

  7. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

    Directory of Open Access Journals (Sweden)

    Nicholas Chia

    Full Text Available BACKGROUND: The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA, replication factor C (RFC, and the minichromosome maintenance (MCM complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. METHODOLOGY/PRINCIPAL FINDINGS: While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. CONCLUSION/SIGNIFICANCE: This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  8. Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Eric A Yen

    2014-05-01

    Full Text Available Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and

  9. Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

    Science.gov (United States)

    Yen, Eric A; Tsay, Aaron; Waldispuhl, Jerome; Vogel, Jackie

    2014-05-01

    Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and visualization of the hidden

  10. Effects of ionizing radiations on DNA-protein complexes; Effets des radiations ionisantes sur des complexes ADN-proteine

    Energy Technology Data Exchange (ETDEWEB)

    Gillard, N

    2005-11-15

    The radio-induced destruction of DNA-protein complexes may have serious consequences for systems implicated in important cellular functions. The first system which has been studied is the lactose operon system, that regulates gene expression in Escherichia coli. First of all, the repressor-operator complex is destroyed after irradiation of the complex or of the protein alone. The damaging of the domain of repressor binding to DNA (headpiece) has been demonstrated and studied from the point of view of peptide chain integrity, conformation and amino acids damages. Secondly, dysfunctions of the in vitro induction of an irradiated repressor-unirradiated DNA complex have been observed. These perturbations, due to a decrease of the number of inducer binding sites, are correlated to the damaging of tryptophan residues. Moreover, the inducer protects the repressor when they are irradiated together, both by acting as a scavenger in the bulk, and by the masking of its binding site on the protein. The second studied system is formed by Fpg (for Formamido pyrimidine glycosylase), a DNA repair protein and a DNA with an oxidative lesion. The results show that irradiation disturbs the repair both by decreasing its efficiency of DNA lesion recognition and binding, and by altering its enzymatic activity. (author)

  11. Measurements of complex refractive indices of photoactive yellow protein

    CERN Document Server

    Lee, KyeoReh; Jung, JaeHwang; Ihee, Hyotcherl; Park, YongKeun

    2015-01-01

    A novel optical technique for measuring the complex refractive index (CRI) of photoactive proteins over the wide range of visible wavelengths is presented. Employing quantitative phase microscopy equipped with a wavelength swept source, optical fields transmitted from a solution of photoactive proteins were precisely measured, from which the CRIs of the photoactive proteins were retrieved with the Fourier light scattering technique. Using the present method, both the real and imaginary RIs of a photoactive yellow protein (PYP) solution were precisely measured over a broad wavelength range (461 - 582 nm). The internal population of the ground and excited states were switched by blue light excitation (445 nm center wavelength), and the broadband refractive index increments of each state were measured. The significant CRI deviation between in the presence and absence of the blue excitation was quantified and explained based on the Kramers-Kronig relations.

  12. Protein-Protein Interaction Investigated by Steered Molecular Dynamics: The TCR-pMHC Complex

    OpenAIRE

    Cuendet, Michel A.; Michielin, Olivier

    2008-01-01

    We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the intera...

  13. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    Science.gov (United States)

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  14. Characterization of the human GARP (Golgi associated retrograde protein) complex

    International Nuclear Information System (INIS)

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells

  15. Changes in protein structure at the interface accompanying complex formation

    Directory of Open Access Journals (Sweden)

    Devlina Chakravarty

    2015-11-01

    Full Text Available Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U and bound (B forms of protein structures from the Protein–Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA, secondary structure, temperature factors (B factors and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69% of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered residues in the U form which are observed (ordered in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial.

  16. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

    Science.gov (United States)

    Bondage, Devanand D; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-07-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  17. A profile of protein-protein interaction: Crystal structure of a lectin-lectin complex.

    Science.gov (United States)

    Surya, Sukumaran; Abhilash, Joseph; Geethanandan, Krishnan; Sadasivan, Chittalakkottu; Haridas, Madhathilkovilakathu

    2016-06-01

    Proteins may utilize complex networks of interactions to create/proceed signaling pathways of highly adaptive responses such as programmed cell death. Direct binary interactions study of proteins may help propose models for protein-protein interaction. Towards this goal we applied a combination of thermodynamic kinetics and crystal structure analyses to elucidate the complexity and diversity in such interactions. By determining the heat change on the association of two galactose-specific legume lectins from Butea monosperma (BML) and Spatholobus parviflorus (SPL) belonging to Fabaceae family helped to compute the binding equilibrium. It was extended further by X-ray structural analysis of BML-SPL binary complex. In order to chart the proteins interacting mainly through their interfaces, identification of the nature of forces which stabilized the association of the lectin-lectin complex was examined. Comprehensive analysis of the BMLSPL complex by isothermal titration calorimetry and X-ray crystal structure threw new light on the lectin-lectin interactions suggesting of their use in diverse areas of glycobiology. PMID:26945504

  18. Immersion freezing of ice nucleation active protein complexes

    Science.gov (United States)

    Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

    2013-06-01

    Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between -5 °C to -38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about -6 °C to about -10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached

  19. Immersion freezing of ice nucleation active protein complexes

    Directory of Open Access Journals (Sweden)

    S. Hartmann

    2013-06-01

    Full Text Available Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS, the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between −5 °C to −38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about −6 °C to about −10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice

  20. Solid-state nanopore detection of protein complexes: applications in healthcare and protein kinetics.

    Science.gov (United States)

    Freedman, Kevin J; Bastian, Arangassery R; Chaiken, Irwin; Kim, Min Jun

    2013-03-11

    Protein conjugation provides a unique look into many biological phenomena and has been used for decades for molecular recognition purposes. In this study, the use of solid-state nanopores for the detection of gp120-associated complexes are investigated. They exhibit monovalent and multivalent binding to anti-gp120 antibody monomer and dimers. In order to investigate the feasibility of many practical applications related to nanopores, detection of specific protein complexes is attempted within a heterogeneous protein sample, and the role of voltage on complexed proteins is researched. It is found that the electric field within the pore can result in unbinding of a freely translocating protein complex within the transient event durations measured experimentally. The strong dependence of the unbinding time with voltage can be used to improve the detection capability of the nanopore system by adding an additional level of specificity that can be probed. These data provide a strong framework for future protein-specific detection schemes, which are shown to be feasible in the realm of a 'real-world' sample and an automated multidimensional method of detecting events.

  1. Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

    Directory of Open Access Journals (Sweden)

    Bunai Christine L

    2009-02-01

    Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

  2. Conformal nanopatterning of extracellular matrix proteins onto topographically complex surfaces.

    Science.gov (United States)

    Sun, Yan; Jallerat, Quentin; Szymanski, John M; Feinberg, Adam W

    2015-02-01

    Our Patterning on Topography (PoT) printing technique enables fibronectin, laminin and other proteins to be applied to biomaterial surfaces in complex geometries that are inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface. Here, we used this method to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment.

  3. How to Build a Complex, Functional Propeller Protein, From Parts.

    Science.gov (United States)

    Clark, Patricia L

    2016-04-01

    By combining ancestral sequence reconstruction and in vitro evolution, Smock et al. identified single motifs that assemble into a functional five-bladed β-propeller, and a likely route for conversion into the more complex, extant single chain fusion. Interestingly, although sequence diversification destabilized five-motif fusions, it also destabilized aggregation-prone intermediates, increasing the level of functional protein in vivo. PMID:26971075

  4. RNA-protein complexes identified by crosslinking of polysomes.

    Science.gov (United States)

    Sköld, S E

    1981-01-01

    The bifunctional cleavable reagent diepoxybutane was used to investigate the crosslinking of proteins to the 16S and 23S RNA in Escherichia coli ribosomes. The crosslinking patterns from polysomes, accumulated in the absence and presence of oxytetracycline, as well as reassociated 70S ribosomes were compared. The 30S proteins: S3, S4, S5, S7, S8, S9, S12, S13, S14 and S18 were recovered crosslinked to the 16S RNA and the 50S: proteins L1, L2, L4, L13, L14-L21, L15, L16, L17, L18-L23, L19-22-24, L27 and L28 were recovered crosslinked to the 23S RNA, in all three associated states. Proteins crosslinked to the RNA of the heterologous subunit and therefore considered to be at or near the ribosomal subunit interface were, for all three states, proteins S1, S4, S6, S9, S12, S13, S14 and S18 from the small subunit and proteins L16, L17, L20 and L27 from the large subunit. Finally, the recovery of intrasubunit crosslinks was measured for the isolated subunits. Additional crosslinked complexes were observed between 16S RNA and S1, S2 as well as S6 from the 30S subunit; and between 23S RNA and L10, L11, L7/12 from the 50S subunit.

  5. The Metastasis-associated Proteins 1 and 2 Form Distinct Protein Complexes with Histone Deacetylase Activity

    Institute of Scientific and Technical Information of China (English)

    Ya-LiYao; Wcn-MingYang

    2005-01-01

    The metastasis-associated protein MTA1 has been shown to express differentially to high levels in metastatic cells. MTA2, which is homologous to MTA1, is a component of the NURD ATP-dependcnt chromatin remodeling and histone deacetylase complex. Here we report evidence that although both human MTA1 and MTA2 repress transcription specifically, are located in the nucleus, and contain associated histone deacetylase activity, they exist in two biochemically distinct protein complexes and may perform different functions pertaining to tumor metastasis. Specifically, both MTA1 and MTA2 complexes exert histone deacetylase activity. However, the MTA1 complex contained HDAC1/2, RbAp46/48, and MBD3, but not Sin3 or Mi2, two important components of the MTA2 complex. Moreover, the MTA2 complex is similar to the HDAC1 complex, suggesting a housekeeping role of the MTA2 complex. The MTA1 complex could be further separated, resulting in acore MTA1-HDAC complex, showing that the histone deacetylase activity and transcriptional repression activity were integral properties of the MTA1 complex. Finally, MTA1, unlike MTA2, did not interact with the pleotropic transcription factor YY1 or the immunophilin FKBP25. We suggest that MTA1 associates with adifferent set of transcription factors from MTA2 and that this property may contribute to the metastatic potential of cells overexpressing MTA1. We also report the finding of human MTA3, which is highly homologous toboth MTA1 and MTA2. However, MTA3 does not repress transcription to a significant level and appears to have a diffused pattern of subcellular localization, suggesting a biological role distinct from that of the other two MTA proteins.

  6. Improved functional overview of protein complexes using inferred epistatic relationships

    LENUS (Irish Health Repository)

    Ryan, Colm

    2011-05-23

    Abstract Background Epistatic Miniarray Profiling(E-MAP) quantifies the net effect on growth rate of disrupting pairs of genes, often producing phenotypes that may be more (negative epistasis) or less (positive epistasis) severe than the phenotype predicted based on single gene disruptions. Epistatic interactions are important for understanding cell biology because they define relationships between individual genes, and between sets of genes involved in biochemical pathways and protein complexes. Each E-MAP screen quantifies the interactions between a logically selected subset of genes (e.g. genes whose products share a common function). Interactions that occur between genes involved in different cellular processes are not as frequently measured, yet these interactions are important for providing an overview of cellular organization. Results We introduce a method for combining overlapping E-MAP screens and inferring new interactions between them. We use this method to infer with high confidence 2,240 new strongly epistatic interactions and 34,469 weakly epistatic or neutral interactions. We show that accuracy of the predicted interactions approaches that of replicate experiments and that, like measured interactions, they are enriched for features such as shared biochemical pathways and knockout phenotypes. We constructed an expanded epistasis map for yeast cell protein complexes and show that our new interactions increase the evidence for previously proposed inter-complex connections, and predict many new links. We validated a number of these in the laboratory, including new interactions linking the SWR-C chromatin modifying complex and the nuclear transport apparatus. Conclusion Overall, our data support a modular model of yeast cell protein network organization and show how prediction methods can considerably extend the information that can be extracted from overlapping E-MAP screens.

  7. Biodegradation of the chitin-protein complex in crustacean cuticle

    Science.gov (United States)

    Artur, Stankiewicz B.; Mastalerz, Maria; Hof, C.H.J.; Bierstedt, A.; Flannery, M.B.; Briggs, D.E.G.; Evershed, R.P.

    1998-01-01

    Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative

  8. Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

    Directory of Open Access Journals (Sweden)

    Françoise Paquet

    Full Text Available In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1 from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

  9. Inferring drug-disease associations based on known protein complexes.

    Science.gov (United States)

    Yu, Liang; Huang, Jianbin; Ma, Zhixin; Zhang, Jing; Zou, Yapeng; Gao, Lin

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html.

  10. Protein complex prediction via verifying and reconstructing the topology of domain-domain interactions

    Directory of Open Access Journals (Sweden)

    Kashima Hisashi

    2010-06-01

    Full Text Available Abstract Background High-throughput methods for detecting protein-protein interactions enable us to obtain large interaction networks, and also allow us to computationally identify the associations of proteins as protein complexes. Although there are methods to extract protein complexes as sets of proteins from interaction networks, the extracted complexes may include false positives because they do not account for the structural limitations of the proteins and thus do not check that the proteins in the extracted complex can simultaneously bind to each other. In addition, there have been few searches for deeper insights into the protein complexes, such as of the topology of the protein-protein interactions or into the domain-domain interactions that mediate the protein interactions. Results Here, we introduce a combinatorial approach for prediction of protein complexes focusing not only on determining member proteins in complexes but also on the DDI/PPI organization of the complexes. Our method analyzes complex candidates predicted by the existing methods. It searches for optimal combinations of domain-domain interactions in the candidates based on an assumption that the proteins in a candidate can form a true protein complex if each of the domains is used by a single protein interaction. This optimization problem was mathematically formulated and solved using binary integer linear programming. By using publicly available sets of yeast protein-protein interactions and domain-domain interactions, we succeeded in extracting protein complex candidates with an accuracy that is twice the average accuracy of the existing methods, MCL, MCODE, or clustering coefficient. Although the configuring parameters for each algorithm resulted in slightly improved precisions, our method always showed better precision for most values of the parameters. Conclusions Our combinatorial approach can provide better accuracy for prediction of protein complexes and also

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

    Science.gov (United States)

    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. PMID:26968364

  12. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

    Science.gov (United States)

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

  13. GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

    Science.gov (United States)

    Thomsen, Alex R B; Plouffe, Bianca; Cahill, Thomas J; Shukla, Arun K; Tarrasch, Jeffrey T; Dosey, Annie M; Kahsai, Alem W; Strachan, Ryan T; Pani, Biswaranjan; Mahoney, Jacob P; Huang, Liyin; Breton, Billy; Heydenreich, Franziska M; Sunahara, Roger K; Skiniotis, Georgios; Bouvier, Michel; Lefkowitz, Robert J

    2016-08-11

    Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs. PMID:27499021

  14. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    International Nuclear Information System (INIS)

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  15. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

    Science.gov (United States)

    Schneider, Tim; Hung, Lee-Hsueh; Schreiner, Silke; Starke, Stefan; Eckhof , Heinrich; Rossbach, Oliver; Reich, Stefan; Medenbach, Jan; Bindereif , Albrecht

    2016-01-01

    Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component. PMID:27510448

  16. Distribution of adenosine deaminase complexing protein (ADCP) in human tissues.

    Science.gov (United States)

    Dinjens, W N; ten Kate, J; van der Linden, E P; Wijnen, J T; Khan, P M; Bosman, F T

    1989-12-01

    The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process. PMID:2573631

  17. Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex.

    Science.gov (United States)

    Dyall, Sabrina D; Yan, Weihong; Delgadillo-Correa, Maria G; Lunceford, Adam; Loo, Joseph A; Clarke, Catherine F; Johnson, Patricia J

    2004-10-28

    Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism. Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an [Fe]-hydrogenase which produces hydrogen. ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria. PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria. Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor. The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear. Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not. Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones. Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues. These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont. PMID:15510149

  18. Inclusion complexes of poly-. beta. -cyclodextrin: a model for pressure effects upon ligand-protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Torgerson, P.M.; Drickamer, H.G.; Weber, G.

    1979-07-10

    Certain protein-ligand complexes are destabilized by application of pressures of the order of 5 to 10 kbar while others are stabilized. This divergent behavior is attributed to differences in compressibility of the protein binding sites. Pressure-stabilized binding is thought by us to be characteristic of soft binding sites, sites in which rotation about backbone bonds permits reduction of the site dimensions under pressure. In contradistinction, hard binding sites do not decrease their size when pressure is applied. As a model for this latter kind we have measured the changes in equilibrium with pressure of complexes of poly-..beta..-cyclodextrin with two fluorescent probes: 8-anilinonaphthalene-1-sulfonate and 6-propionyl-2-(dimethylamino)naphthalene. The standard volume change upon formation of the complexes at 1 atm is similar in both (+9.3 mL/mol), and as expected the incompressibility of the cyclodextrin rings results in a site from which the probes are dissociated by pressure. On the assmption of incompressibility of the binding site, the experimental data permit the calculation of the pressure vs volume curves (compressibility curves) for the probes molecularly dispersed in water. These curves are in broad agreement with those of liquid aliphatic and aromatic hydrocarbons in the low-pressure range (1 to 4 kbar) but indicate a reduced compressibility at the higher pressures. Considerations of relative compressibility offer a quantitative alternative to the usual qualitative discussion of the effects of high pressures upon proteins in terms of the participation of hydrophobic and other bonds.

  19. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

    Science.gov (United States)

    Gell, D; Jackson, S P

    1999-01-01

    In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. PMID:10446239

  20. Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

    CERN Document Server

    Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

    2001-01-01

    Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

  1. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes

    Directory of Open Access Journals (Sweden)

    Jacek Dygut

    2016-10-01

    Full Text Available The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains—In this context we can distinguish: (1 Shared hydrophobic cores (spanning the whole dimer; (2 Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

  2. Hydrophilicity matching - a potential prerequisite for the formation of protein-protein complexes in the cell.

    Directory of Open Access Journals (Sweden)

    Mario Hlevnjak

    Full Text Available A binding event between two proteins typically consists of a diffusional search of binding partners for one another, followed by a specific recognition of the compatible binding sites resulting in the formation of the complex. However, it is unclear how binding partners find each other in the context of the crowded, constantly fluctuating, and interaction-rich cellular environment. Here we examine the non-specific component of protein-protein interactions, which refers to those physicochemical properties of the binding partners that are independent of the exact details of their binding sites, but which can affect their localization or diffusional search for one another. We show that, for a large set of high-resolution experimental 3D structures of binary, transient protein complexes taken from the DOCKGROUND database, the binding partners display a surprising, statistically significant similarity in terms of their total hydration free energies normalized by a size-dependent variable. We hypothesize that colocalization of binding partners, even within individual cellular compartments such as the cytoplasm, may be influenced by their relative hydrophilicity, potentially in response to local hydrophilic gradients.

  3. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

    Science.gov (United States)

    Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

    2016-09-01

    The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  4. Identification of Thylakoid Membrane Protein Complexes by Using a BN-Chip/MS Approach

    Institute of Scientific and Technical Information of China (English)

    Longquan Fan; Yinghong Pan

    2012-01-01

    Thylakoid membrane protein complexes of wheat (Triticum aestivum Linn.)play crucial roles in growth and crop production.Knowledge of the composition and structure of protein complexes,as well as protein interactions,will result in a much deeper understanding of metabolic pathways and cellular processes than protein identities alone,especially if the complexes can be separated in the native forms.Whereas the analysis of membrane protein complexes is a significant challenge due to their hydrophobic properties and relatively low abundance.A rapid and efficient method of identifying membrane protein complexes will greatly facilitate the investigation of agriculture.The present work developed an BN-Chip/MS approach for exhaustive separation and identification of protein complexes,by combining using blue-native polyacrylamide gel electrophoresis (BN-PAGE) and chip-based high-performance liquid chromatography quadruple time-of-flight tandem mass spectrometry (HPLC-Chip/ESI-QT-OF-MS,Chip/MS).By using this approach,seventy-five nonredundant proteins of wheat thylakoid membrane complexes were identified from digested 13 bands of BN-gel.When the protocol of BN separation was not used,only 37 nonredundant proteins had been identified and among of them 9 proteins were uniquely identi? ed.This BN-Chip/MS approach is rapid and efficient for identifying protein complexes in wheat thylakoid membranes,and also providing reliable foundations for further functional research of wheat chloroplast and for identifying protein complexes of other species.

  5. Recovering protein-protein and domain-domain interactions from aggregation of IP-MS proteomics of coregulator complexes.

    Directory of Open Access Journals (Sweden)

    Amin R Mazloom

    2011-12-01

    Full Text Available Coregulator proteins (CoRegs are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP followed by mass spectrometry (MS applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/.

  6. Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Yuichiro Shimizu

    2010-01-01

    Full Text Available We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity of E. coli TDG homolog (EcMUG, which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients.

  7. Piezo dispensed microarray of multivalent chelating thiols for dissecting complex protein-protein interactions.

    Science.gov (United States)

    Klenkar, Goran; Valiokas, Ramûnas; Lundström, Ingemar; Tinazli, Ali; Tampé, Robert; Piehler, Jacob; Liedberg, Bo

    2006-06-01

    The fabrication of a novel biochip, designed for dissection of multiprotein complex formation, is reported. An array of metal chelators has been produced by piezo dispensing of a bis-nitrilotriacetic acid (bis-NTA) thiol on evaporated gold thin films, prestructured with a microcontact printed grid of eicosanethiols. The bis-NTA thiol is mixed in various proportions with an inert, tri(ethylene glycol) hexadecane thiol, and the thickness and morphological homogeneity of the dispensed layers are characterized by imaging ellipsometry before and after back-filling with the same inert thiol and subsequent rinsing. It is found that the dispensed areas display a monotonic increase in thickness with increasing molar fraction of bis-NTA in the dispensing solution, and they are consistently a few Angströms thicker than those prepared at the same molar fraction by solution self-assembly under equilibrium-like conditions. The bulkiness of the bis-NTA tail group and the short period of time available for chemisorption and in-plane organization of the dispensed thiols are most likely responsible for the observed difference in thickness. Moreover, the functional properties of this biochip are demonstrated by studying multiple protein-protein interactions using imaging surface plasmon resonance. The subunits of the type I interferon receptor are immobilized as a composition array determined by the surface concentration of bis-NTA in the array elements. Ligand dissociation kinetics depends on the receptor surface concentration, which is ascribed to the formation of a ternary complex by simultaneous interaction of the ligand with the two receptor subunits. Thus, multiplexed monitoring of binding phenomena at various compositions (receptor densities) offers a powerful tool to dissect protein-protein interactions.

  8. INFLUENCE OF NATURAL ADDITIVES ON PROTEIN COMPLEX OF BREAD

    Directory of Open Access Journals (Sweden)

    Dana Urminská

    2010-11-01

    Full Text Available The study focuses on researching the influence of natural additives on certain technological characteristics of mixtures used for bread production, more particularly the influence of N substances in used raw material on selected qualitative parameters of bread. The blends for bread production to be analysed were prepared by mixing wheat flour with an addition of oat, buckwheat, lentil and chickpea wholegrain flour in different portions (10, 20, 30, 40 and 50 %. The experiment showed that the addition of natural additives worsened the protein complex of the blends used in bread production (worsening also qualitative parameters known as product volume. The loaves prepared with an addition of buckwheat, oat, lentil and chickpea were evaluated to be of a lesser quality from a technological viewpoint when compared with pure wheat loaves. The lower content of gluten forming proteins and the generally changed protein composition of blends due to additives caused a lower percentage of wet gluten content, its lower extensibility and swelling capacity. The sedimentation value (Zeleny index decreased proportionally with the increase of addition until the level was unsatisfactory for raw material intended for bakery purposes. The N content in experimental loaves was higher than in the reference loaves and it increased according to the selected additive and its portion in the blend (more with the addition of lentil and chickpea, less in case of buckwheat and oat which is considered as positive from a nutritional point of view. But from the technological point of view the additives did not show any positive influence and caused a lower loaf bread volume. The most significant decrease of the loaf bread volume was found with the addition of 50 % of buckwheat (- 45.6 %. Better results were obtained with a lower portion of the additive: loaf with an addition of 30 % of chickpea (volume decreased by 12.8 % > loaf with an addition of 30 % of lentil (volume

  9. Study on protein conformation and adsorption behaviors in nanodiamond particle-protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Wang Haidong [Department of Biomedical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Niu, Catherine Hui [Department of Chemical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Yang Qiaoqin [Department of Mechanical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Badea, Ildiko, E-mail: catherine.niu@usask.ca [Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, S7N 5C9 (Canada)

    2011-04-08

    In the present research, the conformation of bovine serum albumin (BSA) in the nanodiamond particle (ND)-BSA complex was studied by Fourier transform infrared spectroscopy, fluorescence spectroscopy, UV-vis spectroscopy, and circular dichroism spectroscopy. The spectroscopic study revealed that most BSA structural features could be preserved in the complex though the BSA underwent conformational changes in the complex due to ND-BSA interaction. In addition, BSA adsorption isotherms and zeta-potential measurements were employed to investigate the pH dependence of the ND-BSA interaction. The changes in surface charge of the ND-BSA complex with pH variations indicated that the binding of BSA to ND might lead to not only the adsorption of BSA onto the ND surface but also the partial breakup of ND aggregates into relatively small ND-BSA aggregates because of the strong binding force between ND and BSA. The results show that ND is an excellent platform for protein immobilization with high affinity and holds great potential to be used for biosensor applications.

  10. Adenosine deaminase complexing protein (ADCP) immunoreactivity in colorectal adenocarcinoma.

    Science.gov (United States)

    ten Kate, J; van den Ingh, H F; Khan, P M; Bosman, F T

    1986-04-15

    Immunoreactive adenosine deaminase complexing protein (ADCP) was studied in 91 human colorectal adenocarcinomas. The expression of ADCP was correlated with that of secretory component (SC) and carcinoembryonic antigen (CEA), with the histological grade and the Dukes' stage of the carcinomas. The histological grade was scored semi-quantitatively according to 5 structural and 4 cytological variables. ADCP expression was observed in 3 different staining patterns, namely: (1) diffuse cytoplasmic (77% of the carcinomas); (2) granular cytoplasmic (13%); and (3) membrane-associated (66%). These patterns were observed alone or in combination. Eleven percent of the carcinomas exhibited no ADCP immunoreactivity. Linear regression analysis showed that the expression of ADCP correlates with that of SC and CEA. However, no significant correlation emerged between the histological parameters or the Dukes' stage and any of the immunohistological parameters. Comparison of the histological characteristics of carcinomas exhibiting little or no ADCP immunoreactivity with those showing extensive immunoreactivity, showed that membranous ADCP immunoreactivity occurs more frequently in well-differentiated carcinomas. Structural parameters showed a better correlation with membranous ADCP expression than the cytological variables. It is concluded that membranous expression of ADCP and CEA are indicators of a high level of differentiation as reflected primarily in the structural characteristics of the tumor. PMID:3957458

  11. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes During In Vitro Pepsin Digestion

    Science.gov (United States)

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated d...

  12. Cationic versus anionic surfactant in tuning the structure and interaction of nanoparticle, protein, and surfactant complexes.

    Science.gov (United States)

    Mehan, Sumit; Aswal, Vinod K; Kohlbrecher, Joachim

    2014-08-26

    The structure and interaction in complexes of anionic Ludox HS40 silica nanoparticle, anionic bovine serum albumin (BSA) protein, and cationic dodecyl trimethylammonium bromide (DTAB) surfactant have been studied using small-angle neutron scattering (SANS). The results are compared with similar complexes having anionic sodium dodecyl sulfate (SDS) surfactant (Mehan, S; Chinchalikar, A. J.; Kumar, S.; Aswal, V. K.; Schweins, R. Langmuir 2013, 29, 11290). In both cases (DTAB and SDS), the structure in nanoparticle-protein-surfactant complexes is predominantly determined by the interactions of the individual two-component systems. The nanoparticle-surfactant (mediated through protein-surfactant complex) and protein-surfactant interactions for DTAB, but nanoparticle-protein (mediated through protein-surfactant complex) and protein-surfactant interactions for SDS, are found to be responsible for the resultant structure of nanoparticle-protein-surfactant complexes. Irrespective of the charge on the surfactant, the cooperative binding of surfactant with protein leads to micellelike clusters of surfactant formed along the unfolded protein chain. The adsorption of these protein-surfactant complexes for DTAB on oppositely charged nanoparticles gives rise to the protein-surfactant complex-mediated aggregation of nanoparticles (similar to that of DTAB surfactant). It is unlike that of depletion-induced aggregation of nanoparticles with nonadsorption of protein-surfactant complexes for SDS in similarly charged nanoparticle systems (similar to that of protein alone). The modifications in nanoparticle aggregation as well as unfolding of protein in these systems as compared to the corresponding two-component systems have also been examined by selectively contrast matching the constituents.

  13. Comparison of label-free quantification methods for the determination of protein complexes subunits stoichiometry

    Directory of Open Access Journals (Sweden)

    Bertrand Fabre

    2014-09-01

    Full Text Available Protein complexes are the main molecular machines that support all major cellular pathways and their in-depth characterization are essential to understand their functions. Determining the stoichiometry of the different subunits of a protein complex still remains challenging. Recently, many label-free quantitative proteomic approaches have been developed to study the composition of protein complexes. It is therefore of great interest to evaluate these different methods in a stoichiometry oriented objective. Here we compare the ability of four absolute quantitative label-free methods currently used in proteomic studies to determine the stoichiometry of a well-characterized protein complex, the 26S proteasome.

  14. Protein complex prediction via improved verification methods using constrained domain-domain matching.

    Science.gov (United States)

    Zhao, Yang; Hayashida, Morihiro; Nacher, Jose C; Nagamochi, Hiroshi; Akutsu, Tatsuya

    2012-01-01

    Identification of protein complexes within protein-protein interaction networks is one of the important objectives in functional genomics. Ozawa et al. proposed a verification method of protein complexes by introducing a structural constraint. In this paper, we propose an improved integer programming-based method based on the idea that a candidate complex should not be divided into many small complexes, and combination methods with maximal components and extreme sets. The results of computational experiments suggest that our methods outperform the method by Ozawa et al. We prove that the verification problems are NP-hard, which justifies the use of integer programming.

  15. A novel phycocyanin-Chla/c2-protein complex isolated from chloroplasts of Chroomonas placoidea

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nine pigment-protein complexes were separated and characterized from intact Chroomonasplacoidea chloroplasts by IEF. The bands Ⅰ-Ⅵ with their isoelectric points (pI) values from 4 to 6 were phycocyanin components; bands Ⅷ and Ⅸ (pI = 2.8-3.6)were chlorophyll-protein complexes. According to absorption and fluorescence spectra, band Ⅶ was designated as a novel phycocyanin-Chla/c2-protein complex (pI ≈ 3.4-3.7). These results indicated that phycocyanin is structurally and functionally coupled with chlorophyll-protein complex in C. placoidea, and probably interacted with electrostatic force in combination.

  16. Qualitative and Quantitative Protein Complex Prediction Through Proteome-Wide Simulations.

    Science.gov (United States)

    Rizzetto, Simone; Priami, Corrado; Csikász-Nagy, Attila

    2015-10-01

    Despite recent progress in proteomics most protein complexes are still unknown. Identification of these complexes will help us understand cellular regulatory mechanisms and support development of new drugs. Therefore it is really important to establish detailed information about the composition and the abundance of protein complexes but existing algorithms can only give qualitative predictions. Herein, we propose a new approach based on stochastic simulations of protein complex formation that integrates multi-source data--such as protein abundances, domain-domain interactions and functional annotations--to predict alternative forms of protein complexes together with their abundances. This method, called SiComPre (Simulation based Complex Prediction), achieves better qualitative prediction of yeast and human protein complexes than existing methods and is the first to predict protein complex abundances. Furthermore, we show that SiComPre can be used to predict complexome changes upon drug treatment with the example of bortezomib. SiComPre is the first method to produce quantitative predictions on the abundance of molecular complexes while performing the best qualitative predictions. With new data on tissue specific protein complexes becoming available SiComPre will be able to predict qualitative and quantitative differences in the complexome in various tissue types and under various conditions.

  17. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry

    DEFF Research Database (Denmark)

    Ho, Yuen; Gruhler, Albrecht; Heilbut, Adrian;

    2002-01-01

    as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were......The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects...... binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae...

  18. Supervised maximum-likelihood weighting of composite protein networks for complex prediction

    Directory of Open Access Journals (Sweden)

    Yong Chern Han

    2012-12-01

    Full Text Available Abstract Background Protein complexes participate in many important cellular functions, so finding the set of existent complexes is essential for understanding the organization and regulation of processes in the cell. With the availability of large amounts of high-throughput protein-protein interaction (PPI data, many algorithms have been proposed to discover protein complexes from PPI networks. However, such approaches are hindered by the high rate of noise in high-throughput PPI data, including spurious and missing interactions. Furthermore, many transient interactions are detected between proteins that are not from the same complex, while not all proteins from the same complex may actually interact. As a result, predicted complexes often do not match true complexes well, and many true complexes go undetected. Results We address these challenges by integrating PPI data with other heterogeneous data sources to construct a composite protein network, and using a supervised maximum-likelihood approach to weight each edge based on its posterior probability of belonging to a complex. We then use six different clustering algorithms, and an aggregative clustering strategy, to discover complexes in the weighted network. We test our method on Saccharomyces cerevisiae and Homo sapiens, and show that complex discovery is improved: compared to previously proposed supervised and unsupervised weighting approaches, our method recalls more known complexes, achieves higher precision at all recall levels, and generates novel complexes of greater functional similarity. Furthermore, our maximum-likelihood approach allows learned parameters to be used to visualize and evaluate the evidence of novel predictions, aiding human judgment of their credibility. Conclusions Our approach integrates multiple data sources with supervised learning to create a weighted composite protein network, and uses six clustering algorithms with an aggregative clustering strategy to

  19. Observation of two different fractal structures in nanoparticle, protein and surfactant complexes

    Energy Technology Data Exchange (ETDEWEB)

    Mehan, Sumit, E-mail: sumit.mehan@gmail.com; Kumar, Sugam, E-mail: sumit.mehan@gmail.com; Aswal, V. K., E-mail: sumit.mehan@gmail.com [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai-400085 (India)

    2014-04-24

    Small angle neutron scattering has been carried out from a complex of nanoparticle, protein and surfactant. Although all the components are similarly (anionic) charged, we have observed strong interactions in their complex formation. It is characterized by the coexistence of two different mass fractal structures. The first fractal structure is originated from the protein and surfactant interaction and second from the depletion effect of first fractal structure leading the nanoparticle aggregation. The fractal structure of protein-surfactant complex represents to bead necklace structure of micelle-like clusters of surfactant formed along the unfolded protein chain. Its fractal dimension depends on the surfactant to protein ratio (r) and decreases with the increase in r. However, fractal dimension of nanoparticle aggregates in nanoparticle-protein complex is found to be independent of protein concentration and governed by the diffusion limited aggregation like morphology.

  20. Observation of two different fractal structures in nanoparticle, protein and surfactant complexes

    Science.gov (United States)

    Mehan, Sumit; Kumar, Sugam; Aswal, V. K.

    2014-04-01

    Small angle neutron scattering has been carried out from a complex of nanoparticle, protein and surfactant. Although all the components are similarly (anionic) charged, we have observed strong interactions in their complex formation. It is characterized by the coexistence of two different mass fractal structures. The first fractal structure is originated from the protein and surfactant interaction and second from the depletion effect of first fractal structure leading the nanoparticle aggregation. The fractal structure of protein-surfactant complex represents to bead necklace structure of micelle-like clusters of surfactant formed along the unfolded protein chain. Its fractal dimension depends on the surfactant to protein ratio (r) and decreases with the increase in r. However, fractal dimension of nanoparticle aggregates in nanoparticle-protein complex is found to be independent of protein concentration and governed by the diffusion limited aggregation like morphology.

  1. Analysis of protein-protein docking decoys using interaction fingerprints: application to the reconstruction of CaM-ligand complexes

    Directory of Open Access Journals (Sweden)

    Uchikoga Nobuyuki

    2010-05-01

    Full Text Available Abstract Background Protein-protein docking for proteins with large conformational changes was analyzed by using interaction fingerprints, one of the scales for measuring similarities among complex structures, utilized especially for searching near-native protein-ligand or protein-protein complex structures. Here, we have proposed a combined method for analyzing protein-protein docking by taking large conformational changes into consideration. This combined method consists of ensemble soft docking with multiple protein structures, refinement of complexes, and cluster analysis using interaction fingerprints and energy profiles. Results To test for the applicability of this combined method, various CaM-ligand complexes were reconstructed from the NMR structures of unbound CaM. For the purpose of reconstruction, we used three known CaM-ligands, namely, the CaM-binding peptides of cyclic nucleotide gateway (CNG, CaM kinase kinase (CaMKK and the plasma membrane Ca2+ ATPase pump (PMCA, and thirty-one structurally diverse CaM conformations. For each ligand, 62000 CaM-ligand complexes were generated in the docking step and the relationship between their energy profiles and structural similarities to the native complex were analyzed using interaction fingerprint and RMSD. Near-native clusters were obtained in the case of CNG and CaMKK. Conclusions The interaction fingerprint method discriminated near-native structures better than the RMSD method in cluster analysis. We showed that a combined method that includes the interaction fingerprint is very useful for protein-protein docking analysis of certain cases.

  2. Nuclear domain 10-associated proteins recognize and segregate intranuclear DNA/protein complexes to negate gene expression

    Directory of Open Access Journals (Sweden)

    Rivera-Molina Yisel A

    2012-09-01

    Full Text Available Abstract Background DNA viruses, such as herpes simplex virus type 1 (HSV-1, Simian virus 40 (SV40, and Cytomegaloviruses (CMV, start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies. It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. Results Our recent observations provide evidence that a foreign (not endogenous DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor, or from HPV11 (human papillomavirus 11 origin DNA and its binding protein (E2, co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA showed no association with ND10. Furthermore, our examination of PML−/−, Daxx−/−, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. Conclusion Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.

  3. Toluene 4-Monooxygenase and its Complex with Effector Protein T4moD

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, Lucas J.; Fox, Brian G. (UW)

    2012-10-16

    Toluene 4-monooxygenase (T4MO) is a multiprotein diiron enzyme complex that catalyzes the regiospecific oxidation of toluene to p-cresol. Catalytic function requires the presence of a small protein, called the effector protein. Effector protein exerts substantial control on the diiron hydroxylase catalytic cycle through protein-protein interactions. High-resolution crystal structures of the stoichometric hydroxylase and effector protein complex described here reveal how protein-protein interactions and reduction of the diiron center produce an active site configuration poised for reaction with O{sub 2}. Further information from crystal structures of mutated isoforms of the hydroxylase and a peroxo adduct is combined with catalytic results to give a fuller picture of the geometry of the enzyme-substrate complex used for the high fidelity oxidation of hydrocarbon substrates.

  4. Kinetics of protein-protein complex coacervation and biphasic release of salbutamol sulfate from coacervate matrix.

    Science.gov (United States)

    Tiwari, Ananya; Bindal, Sonal; Bohidar, H B

    2009-01-12

    Turbidimetric titration was used to initiate associative intermolecular interactions between a pair of protein molecules, gelatin-A and gelatin-B, having complementary charges that led to pH-induced liquid-liquid phase separation and the formation of complex coacervate. The stoichiometric binding ratio was found to be [gelatin-A]/[gelatin-B]=3:2. The size of soluble intermolecular aggregates present in the supernatant exhibited interesting time-dependent coacervation because of residual electrostatic interactions. Dynamic light scattering and turbidity studies provided a systematic account of coacervation behavior. Rheology studies attributed the softening of the coacervate matrix to the presence of encapsulated salbutamol sulfate. The in vitro drug release kinetics was probed in simulated gastric fluid medium at physiological temperature (37 degrees C), which showed biphasic behavior. The initial release kinetics exhibited an exponential growth to saturation behavior, followed by a slower logarithmic release process.

  5. Adenosine deaminase complexing protein (ADCP): a transformation sensitive protein with potentials of a cancer marker.

    Science.gov (United States)

    Herbschleb-Voogt, E; Ten Kate, J; Meera Khan, P

    1983-01-01

    Several observations by independent investigators in the past have indicated that adenosine deaminase complexing protein (ADCP), present in considerable quantities in certain human tissues, was absent or decreased in the cancers originated from them. During the present study, electrophoretic analysis of adenosine deaminase (ADA) isozymes and radioimmunoassay for ADCP in the primary fibroblasts and the transformed as well as certain tumor derived cell lines have demonstrated that ADCP present in large quantities in the primary cells was absent or nearly absent in the transformed or tumor-derived cell lines. Though the mechanisms involved are not yet clear, the above observations indicate that ADCP has the potentials of a useful marker in the studies on transformed cells and cancer tissues. PMID:6133497

  6. BISC: Binary SubComplexes in proteins database

    OpenAIRE

    Juettemann, Thomas; Gerloff, Dietlind L

    2010-01-01

    Binary subcomplexes in proteins database (BISC) is a new protein–protein interaction (PPI) database linking up the two communities most active in their characterization: structural biology and functional genomics researchers. The BISC resource offers users (i) a structural perspective and related information about binary subcomplexes (i.e. physical direct interactions between proteins) that are either structurally characterized or modellable entries in the main functional genomics PPI databas...

  7. Gold Nanocluster and Quantum Dot Complex in Protein for Biofriendly White-Light-Emitting Material.

    Science.gov (United States)

    Bhandari, Satyapriya; Pramanik, Sabyasachi; Khandelia, Rumi; Chattopadhyay, Arun

    2016-01-27

    We report the synthesis of a biofriendly highly luminescent white-light-emitting nanocomposite. The composite consisted of Au nanoclusters and ZnQ2 complex (on the surface of ZnS quantum dots) embedded in protein. The combination of red, green, and blue luminescence from clusters, complex, and protein, respectively, led to white light generation. PMID:26741861

  8. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  9. Biophysical characterization of the complex between human papillomavirus E6 protein and synapse-associated protein 97

    DEFF Research Database (Denmark)

    Chi, Celestine Ngang; Bach, Anders; Engström, Åke;

    2011-01-01

    The E6 protein of human papillomavirus exhibits complex interaction patterns with several host proteins and their roles in HPV mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppr...

  10. iTreePack: Protein Complex Side-Chain Packing by Dual Decomposition

    OpenAIRE

    Peng, Jian; Hosur, Raghavendra; Berger, Bonnie; Xu, Jinbo

    2015-01-01

    Protein side-chain packing is a critical component in obtaining the 3D coordinates of a structure and drug discovery. Single-domain protein side-chain packing has been thoroughly studied. A major challenge in generalizing these methods to protein complexes is that they, unlike monomers, often have very large treewidth, and thus algorithms such as TreePack cannot be directly applied. To address this issue, SCWRL4 treats the complex effectively as a monomer, heuristically excluding weak interac...

  11. Interaction proteomics analysis of polycomb proteins defines distinct PRC1 complexes in mammalian cells

    DEFF Research Database (Denmark)

    Vandamme, Julien; Völkel, Pamela; Rosnoblet, Claire;

    2011-01-01

    Polycomb group (PcG) proteins maintain transcriptional repression of hundreds of genes involved in development, signaling or cancer using chromatin-based epigenetic mechanisms. Biochemical studies in Drosophila have revealed that PcG proteins associate in at least two classes of protein complexes...

  12. Counterions release from electrostatic complexes of polyelectrolytes and proteins of opposite charge : a direct measurement

    OpenAIRE

    Gummel, Jérémie; Cousin, Fabrice; Boué, François

    2009-01-01

    Though often considered as one of the main driving process of the complexation of species of opposite charges, the release of counterions has never been experimentally directly measured on polyelectrolyte/proteins complexes. We present here the first structural determination of such a release by Small Angle Neutron Scattering in complexes made of lysozyme, a positively charged protein and of PSS, a negatively charged polyelectrolyte. Both components have the same neutron density length, so th...

  13. A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Fan Keqiang

    2011-01-01

    Full Text Available Abstract Background Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology. Results A two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation. Conclusion Two dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes.

  14. Understanding the nanoparticle-protein corona complexes using computational and experimental methods.

    Science.gov (United States)

    Kharazian, B; Hadipour, N L; Ejtehadi, M R

    2016-06-01

    Nanoparticles (NP) have capability to adsorb proteins from biological fluids and form protein layer, which is called protein corona. As the cell sees corona coated NPs, the protein corona can dictate biological response to NPs. The composition of protein corona is varied by physicochemical properties of NPs including size, shape, surface chemistry. Processing of protein adsorption is dynamic phenomena; to that end, a protein may desorb or leave a surface vacancy that is rapidly filled by another protein and cause changes in the corona composition mainly by the Vroman effect. In this review, we discuss the interaction between NP and proteins and the available techniques for identification of NP-bound proteins. Also we review current developed computational methods for understanding the NP-protein complex interactions.

  15. Evidence for the robustness of protein complexes to inter-species hybridization.

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Leducq

    Full Text Available Despite the tremendous efforts devoted to the identification of genetic incompatibilities underlying hybrid sterility and inviability, little is known about the effect of inter-species hybridization at the protein interactome level. Here, we develop a screening platform for the comparison of protein-protein interactions (PPIs among closely related species and their hybrids. We examine in vivo the architecture of protein complexes in two yeast species (Saccharomyces cerevisiae and Saccharomyces kudriavzevii that diverged 5-20 million years ago and in their F1 hybrids. We focus on 24 proteins of two large complexes: the RNA polymerase II and the nuclear pore complex (NPC, which show contrasting patterns of molecular evolution. We found that, with the exception of one PPI in the NPC sub-complex, PPIs were highly conserved between species, regardless of protein divergence. Unexpectedly, we found that the architecture of the complexes in F1 hybrids could not be distinguished from that of the parental species. Our results suggest that the conservation of PPIs in hybrids likely results from the slow evolution taking place on the very few protein residues involved in the interaction or that protein complexes are inherently robust and may accommodate protein divergence up to the level that is observed among closely related species.

  16. A Novel Algorithm for Detecting Protein Complexes with the Breadth First Search

    Directory of Open Access Journals (Sweden)

    Xiwei Tang

    2014-01-01

    Full Text Available Most biological processes are carried out by protein complexes. A substantial number of false positives of the protein-protein interaction (PPI data can compromise the utility of the datasets for complexes reconstruction. In order to reduce the impact of such discrepancies, a number of data integration and affinity scoring schemes have been devised. The methods encode the reliabilities (confidence of physical interactions between pairs of proteins. The challenge now is to identify novel and meaningful protein complexes from the weighted PPI network. To address this problem, a novel protein complex mining algorithm ClusterBFS (Cluster with Breadth-First Search is proposed. Based on the weighted density, ClusterBFS detects protein complexes of the weighted network by the breadth first search algorithm, which originates from a given seed protein used as starting-point. The experimental results show that ClusterBFS performs significantly better than the other computational approaches in terms of the identification of protein complexes.

  17. Native gel analysis of macromolecular protein complexes in cultured mammalian cells.

    Science.gov (United States)

    Munawar, Nayla; Olivero, Giorgio; Jerman, Emilia; Doyle, Benjamin; Streubel, Gundula; Wynne, Kieran; Bracken, Adrian; Cagney, Gerard

    2015-11-01

    Native gel electrophoresis enables separation of cellular proteins in their non-denatured state. In experiments aimed at analysing proteins in higher order or multimeric assemblies (i.e. protein complexes) it offers some advantages over rival approaches, particularly as an interface technology with mass spectrometry. Here we separated fractions from HEK293 cells by native electrophoresis in order to survey protein complexes in the cytoplasmic, nuclear and chromatin environments, finding 689 proteins distributed among 217 previously described complexes. As expected, different fractions contained distinct combinations of macromolecular complexes, with subunits of the same complex tending to co-migrate. Exceptions to this observation could often be explained by the presence of subunits shared among different complexes. We investigated one identified complex, the Polycomb Repressor Complex 2 (PRC2), in more detail following affinity purification of the EZH2 subunit. This approach resulted in the identification of all previously reported members of PRC2. Overall, this work demonstrates that the use of native gel electrophoresis as an upstream separating step is an effective approach for analysis of the components and cellular distribution of protein complexes. PMID:26223664

  18. PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes.

    Science.gov (United States)

    Han, Kyungsook; Nepal, Chirag

    2007-05-01

    A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/.

  19. A novel approach to analyze membrane proteins by laser mass spectrometry: from protein subunits to the integral complex.

    Science.gov (United States)

    Morgner, Nina; Kleinschroth, Thomas; Barth, Hans-Dieter; Ludwig, Bernd; Brutschy, Bernhard

    2007-08-01

    A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices. PMID:17544294

  20. Structural insights into yeast histone chaperone Hif1: a scaffold protein recruiting protein complexes to core histones.

    Science.gov (United States)

    Liu, Hejun; Zhang, Mengying; He, Wei; Zhu, Zhongliang; Teng, Maikun; Gao, Yongxiang; Niu, Liwen

    2014-09-15

    Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

  1. Tyrosine-selective protein alkylation using pi-allylpalladium complexes.

    Science.gov (United States)

    Tilley, S David; Francis, Matthew B

    2006-02-01

    A new protein modification reaction has been developed based on a palladium-catalyzed allylic alkylation of tyrosine residues. This technique employs electrophilic pi-allyl intermediates derived from allylic acetate and carbamate precursors and can be used to modify proteins in aqueous solution at room temperature. To facilitate the detection of modified proteins using SDS-PAGE analysis, a fluorescent allyl acetate was synthesized and coupled to chymotrypsinogen A and bacteriophage MS2. The tyrosine selectivity of the reaction was confirmed through trypsin digest analysis. The utility of the reaction was demonstrated by using taurine-derived carbamates as water solubilizing groups that are cleaved upon protein functionalization. This solubility switching technique was used to install hydrophobic farnesyl and C(17) chains on chymotrypsinogen A in water using little or no cosolvent. Following this, the C(17) alkylated proteins were found to associate with lipid vesicles. In addition to providing a new protein modification strategy targeting an under-utilized amino acid side chain, this method provides convenient access to synthetic lipoproteins. PMID:16433516

  2. On the interconnection of stable protein complexes: inter-complex hubs and their conservation in Saccharomyces cerevisiae and Homo sapiens networks.

    Science.gov (United States)

    Guerra, Concettina

    2015-01-01

    Protein complexes are key molecular entities that perform a variety of essential cellular functions. The connectivity of proteins within a complex has been widely investigated with both experimental and computational techniques. We developed a computational approach to identify and characterise proteins that play a role in interconnecting complexes. We computed a measure of inter-complex centrality, the crossroad index, based on disjoint paths connecting proteins in distinct complexes and identified inter-complex hubs as proteins with a high value of the crossroad index. We applied the approach to a set of stable complexes in Saccharomyces cerevisiae and in Homo sapiens. Just as done for hubs, we evaluated the topological and biological properties of inter-complex hubs addressing the following questions. Do inter-complex hubs tend to be evolutionary conserved? What is the relation between crossroad index and essentiality? We found a good correlation between inter-complex hubs and both evolutionary conservation and essentiality.

  3. Comparative evolutionary analysis of protein complexes in E. coli and yeast

    Directory of Open Access Journals (Sweden)

    Ranea Juan AG

    2010-02-01

    Full Text Available Abstract Background Proteins do not act in isolation; they frequently act together in protein complexes to carry out concerted cellular functions. The evolution of complexes is poorly understood, especially in organisms other than yeast, where little experimental data has been available. Results We generated accurate, high coverage datasets of protein complexes for E. coli and yeast in order to study differences in the evolution of complexes between these two species. We show that substantial differences exist in how complexes have evolved between these organisms. A previously proposed model of complex evolution identified complexes with cores of interacting homologues. We support findings of the relative importance of this mode of evolution in yeast, but find that it is much less common in E. coli. Additionally it is shown that those homologues which do cluster in complexes are involved in eukaryote-specific functions. Furthermore we identify correlated pairs of non-homologous domains which occur in multiple protein complexes. These were identified in both yeast and E. coli and we present evidence that these too may represent complex cores in yeast but not those of E. coli. Conclusions Our results suggest that there are differences in the way protein complexes have evolved in E. coli and yeast. Whereas some yeast complexes have evolved by recruiting paralogues, this is not apparent in E. coli. Furthermore, such complexes are involved in eukaryotic-specific functions. This implies that the increase in gene family sizes seen in eukaryotes in part reflects multiple family members being used within complexes. However, in general, in both E. coli and yeast, homologous domains are used in different complexes.

  4. Injectable, thermo-reversible and complex coacervate combination gels for protein drug delivery.

    Science.gov (United States)

    Jin, Kwang-Mi; Kim, Yong-Hee

    2008-05-01

    Injectable and thermo-reversible physical combination gels were formed in aqueous solution by preparing complex coacervate with two oppositely charged biomacromolecules that composed of negatively charged chondroitin 6-sulfate and positively charged high molecular weight gelatin type A and co-formulating with a negative, thermo-sensitive polysaccharide, methylcellulose containing a salting-out salt, ammonium sulfate. The combination of complex coacervation and a thermo-reversible gel demonstrated synergistic effects on the complex coacervate formation the release rates of model proteins and in situ gel depot formation. Gels indicated sustained release patterns of the protein over 25 days with minimal initial bursts. Optimized novel in situ gel depot systems containing dual advantages of complex coacervation and temperature responsiveness demonstrated a potential for efficient protein drug delivery in terms of high protein loading, sustained protein release, ease of administration, an aqueous environment without toxic organic solvents, and a simple fabrication method.

  5. Monte Carlo simulations of flexible polyanions complexing with whey proteins at their isoelectric point

    Science.gov (United States)

    de Vries, R.

    2004-02-01

    Electrostatic complexation of flexible polyanions with the whey proteins α-lactalbumin and β-lactoglobulin is studied using Monte Carlo simulations. The proteins are considered at their respective isoelectric points. Discrete charges on the model polyelectrolytes and proteins interact through Debye-Hückel potentials. Protein excluded volume is taken into account through a coarse-grained model of the protein shape. Consistent with experimental results, it is found that α-lactalbumin complexes much more strongly than β-lactoglobulin. For α-lactalbumin, strong complexation is due to localized binding to a single large positive "charge patch," whereas for β-lactoglobulin, weak complexation is due to diffuse binding to multiple smaller charge patches.

  6. Application of linker technique to trap transiently interacting protein complexes for structural studies

    OpenAIRE

    Reddy Chichili, Vishnu Priyanka; Kumar, Veerendra; J Sivaraman

    2016-01-01

    Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational ana...

  7. Capture of unstable protein complex on the streptavidin-coated single-walled carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Liu Zunfeng, E-mail: liuz2@chem.leidenuniv.nl; Voskamp, Patrick [Cell Observatory, Biophysical Structural Chemistry, Leiden Institute of Chemistry (Netherlands); Zhang Yue; Chu Fuqiang [Changzhou University, School of Pharmaceutical Engineering and Life Science (China); Abrahams, Jan Pieter, E-mail: abrahams@chem.leidenuniv.nl [Cell Observatory, Biophysical Structural Chemistry, Leiden Institute of Chemistry (Netherlands)

    2013-04-15

    Purification of unstable protein complexes is a bottleneck for investigation of their 3D structure and in protein-protein interaction studies. In this paper, we demonstrate that streptavidin-coated single-walled carbon nanotubes (Strep Bullet SWNT) can be used to capture the biotinylated DNA-EcoRI complexes on a 2D surface and in solution using atomic force microscopy and electrophoresis analysis, respectively. The restriction enzyme EcoRI forms unstable complexes with DNA in the absence of Mg{sup 2+}. Capturing the EcoRI-DNA complexes on the Strep Bullet SWNT succeeded in the absence of Mg{sup 2+}, demonstrating that the Strep Bullet SWNT can be used for purifying unstable protein complexes.

  8. Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

    Science.gov (United States)

    Gubbens, Jacob; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2008-12-01

    Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins. PMID:18817900

  9. Chaperonin Structure - The Large Multi-Subunit Protein Complex

    Directory of Open Access Journals (Sweden)

    Irena Roterman

    2009-03-01

    Full Text Available The multi sub-unit protein structure representing the chaperonins group is analyzed with respect to its hydrophobicity distribution. The proteins of this group assist protein folding supported by ATP. The specific axial symmetry GroEL structure (two rings of seven units stacked back to back - 524 aa each and the GroES (single ring of seven units - 97 aa each polypeptide chains are analyzed using the hydrophobicity distribution expressed as excess/deficiency all over the molecule to search for structure-to-function relationships. The empirically observed distribution of hydrophobic residues is confronted with the theoretical one representing the idealized hydrophobic core with hydrophilic residues exposure on the surface. The observed discrepancy between these two distributions seems to be aim-oriented, determining the structure-to-function relation. The hydrophobic force field structure generated by the chaperonin capsule is presented. Its possible influence on substrate folding is suggested.

  10. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    Science.gov (United States)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  11. Carotenoid-protein complexes and their stability towards oxygen and radiation

    International Nuclear Information System (INIS)

    Carotenoid-protein complexes isolated from fresh mangoes were found to be more stable to oxygen and radiation when dissolved in water as compared with β-carotene in petroleum ether. Part of the pigment could be released from the complex by gamma irradiation. Observations on the stability of the carotenoid (98% β-carotene) in the complex indicated that the pigment is either associated with the lipid prosthetic group of the protein or loosely attached to the protein by weak hydrophobic bonds. (author)

  12. Acute phase proteins in cattle after exposure to complex stress

    DEFF Research Database (Denmark)

    Lomborg, S. R.; Nielsen, L. R.; Heegaard, Peter M. H.;

    2008-01-01

    Abstract Stressors such as weaning, mixing and transportation have been shown to lead to increased blood concentrations of acute phase proteins (APP), including serum amyloid A (SAA) and haptoglobin, in calves. This study was therefore undertaken to assess whether SAA and haptoglobin levels...

  13. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    Science.gov (United States)

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1. PMID:27235398

  14. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    Science.gov (United States)

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1.

  15. Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.

    Science.gov (United States)

    Money, T; Jones, T; Dixon, R; Austin, S

    1999-08-01

    In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation. PMID:10419940

  16. Stability and immunogenicity of hypoallergenic peanut protein-polyphenol complexes during in vitro pepsin digestion.

    Science.gov (United States)

    Plundrich, Nathalie J; White, Brittany L; Dean, Lisa L; Davis, Jack P; Foegeding, E Allen; Lila, Mary Ann

    2015-07-01

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated during simulated in vitro gastric digestion. When digested with pepsin, the basic subunit of the peanut allergen Ara h 3 was more rapidly hydrolyzed in peanut protein-cranberry or green tea polyphenol complexes compared to uncomplexed peanut flour. Ara h 2 was also hydrolyzed more quickly in the peanut protein-cranberry polyphenol complex than in uncomplexed peanut flour. Peptides from peanut protein-cranberry polyphenol complexes and peanut protein-green tea polyphenol complexes were substantially less immunoreactive (based on their capacity to bind to peanut-specific IgE from patient plasma) compared to peptides from uncomplexed peanut flour. These results suggest that peanut protein-polyphenol complexes may be less immunoreactive passing through the digestive tract in vivo, contributing to their attenuated allergenicity.

  17. Exposing the subunit diversity within protein complexes: a mass spectrometry approach.

    Science.gov (United States)

    Rozen, Shelly; Tieri, Alessandra; Ridner, Gabriela; Stark, Ann-Kathrin; Schmaler, Tilo; Ben-Nissan, Gili; Dubiel, Wolfgang; Sharon, Michal

    2013-03-01

    Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge alone does not reveal the full complexity of protein assemblies, as each subunit can take on multiple forms. Proteins can be post-translationally modified or cleaved, multiple products of alternative splicing can exist, and a single subunit may be encoded by more than one gene. Thus, for a complete description of a protein complex, it is necessary to expose the diversity of its subunits. Adding this layer of information is an important step towards understanding the mechanisms that regulate the activity of protein assemblies. Here, we describe a mass spectrometry-based approach that exposes the array of protein variants that comprise protein complexes. Our method relies on denaturing the protein complex, and separating its constituent subunits on a monolithic column prepared in-house. Following the subunit elution from the column, the flow is split into two fractions, using a Triversa NanoMate robot. One fraction is directed straight into an on-line ESI-QToF mass spectrometer for intact protein mass measurements, while the rest of the flow is fractionated into a 96-well plate for subsequent proteomic analysis. The heterogeneity of subunit composition is then exposed by correlating the subunit sequence identity with the accurate mass. Below, we describe in detail the methodological setting of this approach, its application on the endogenous human COP9 signalosome complex, and the significance of the method for structural mass spectrometry analysis of intact protein complexes. PMID:23296018

  18. Supramolecular complexes of the Agrobacterium tumefaciens virulence protein VirE2.

    Science.gov (United States)

    Volokhina, I V; Gusev, Yu S; Mazilov, S I; Chumakov, M I

    2011-11-01

    Virulence protein VirE2 from Agrobacterium tumefaciens is involved in plant infection by transferring a fragment of agrobacterial Ti plasmid ssT-DNA in complex with VirE2-VirD2 proteins into the plant cell nucleus. The VirE2 protein interactions with ssDNA and formation of VirE2 protein complexes in vitro and in silico have been studied. Using dynamic light scattering we found that purified recombinant protein VirE2 exists in buffer solution in the form of complexes of 2-4 protein molecules of 12-18 nm size. We used computer methods to design models of complexes consisting of two and four individual VirE2 proteins, and their dimensions were estimated. Dimensions of VirE2 complexes with ssDNA (550 and 700 nucleotide residues) were determined using transmission electron microscopy and dynamic light scattering. We found that in vitro, upon interaction with ssDNA recombinant protein, VirE2 is able to alter conformation of the latter by shortening the initial length of the ssDNA. PMID:22117554

  19. Identification of protein complexes required for efficient sister chromatid cohesion

    NARCIS (Netherlands)

    Mayer, Melanie L; Pot, Isabelle; Chang, Michael; Xu, Hong; Aneliunas, Victoria; Kwok, Teresa; Newitt, Rick; Aebersold, Ruedi; Boone, Charles; Brown, Grant W; Hieter, Philip

    2004-01-01

    Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes r

  20. The challenges in and importance of analysing protein structure and physical stability in complex formulations

    DEFF Research Database (Denmark)

    Jorgensen, L.; Jensen, Minna Grønning; Roest, N.;

    2013-01-01

    In this review several analytical challenges that may be encountered during protein formulation development of complex formulations are discussed through recent examples. These examples show how selected advanced biophysical methods can greatly increase our understanding of the system under inves...

  1. Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

    DEFF Research Database (Denmark)

    Selao, Tiago Toscano; Branca, Rui; Chae, Pil Seok;

    2011-01-01

    The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the u...

  2. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    Science.gov (United States)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  3. Protein complexes and cholesterol in the control of late endosomal dynamicsCholesterol and multi-protein complexes in the control of late endosomal dynamics

    NARCIS (Netherlands)

    Kant, Rik Henricus Nicolaas van der

    2013-01-01

    Late endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer’s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the

  4. Database of amino acid-nucleotide contacts in the DNA complexes with homeodomain family proteins

    International Nuclear Information System (INIS)

    The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires analysis of the physicochemical characteristics of these contacts, of the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used for comparison and classification of interfaces of the protein-DNA complexes

  5. The beginning of a beautiful friendship: Cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes

    Science.gov (United States)

    Rappsilber, Juri

    2011-01-01

    After more than a decade of method development, cross-linking in combination with mass spectrometry and bioinformatics is finally coming of age. This technology now provides improved opportunities for modelling by mapping structural details of functional complexes in solution. The structure of proteins or protein complexes is ascertained by identifying amino acid pairs that are positioned in close proximity to each other. The validity of this technique has recently been benchmarked for large multi-protein complexes, by comparing cross-link data with that from a crystal structure of RNA polymerase II. Here, the specific nature of this cross-linking data will be discussed to assess the technical challenges and opportunities for model building. We believe that once remaining technological challenges of cross-linking/mass spectrometry have been addressed and cross-linking/mass spectrometry data has been incorporated into modelling algorithms it will quickly become an indispensable companion of protein and protein complex modelling and a corner-stone of integrated structural biology. PMID:21029779

  6. Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex.

    OpenAIRE

    Okada, T; Ramsey, W J; Munir, J; Wildner, O.; Blaese, R M

    1998-01-01

    We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promo...

  7. On the importance of polar interactions for complexes containing intrinsically disordered proteins.

    Directory of Open Access Journals (Sweden)

    Eric T C Wong

    Full Text Available There is a growing recognition for the importance of proteins with large intrinsically disordered (ID segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.

  8. Conservation and Variability of Synaptonemal Complex Proteins in Phylogenesis of Eukaryotes

    Directory of Open Access Journals (Sweden)

    Tatiana M. Grishaeva

    2014-01-01

    Full Text Available The problems of the origin and evolution of meiosis include the enigmatic variability of the synaptonemal complexes (SCs which, being morphology similar, consist of different proteins in different eukaryotic phyla. Using bioinformatics methods, we monitored all available eukaryotic proteomes to find proteins similar to known SC proteins of model organisms. We found proteins similar to SC lateral element (LE proteins and possessing the HORMA domain in the majority of the eukaryotic taxa and assume them the most ancient among all SC proteins. Vertebrate LE proteins SYCP2, SYCP3, and SC65 proved to have related proteins in many invertebrate taxa. Proteins of SC central space are most evolutionarily variable. It means that different protein-protein interactions can exist to connect LEs. Proteins similar to the known SC proteins were not found in Euglenophyta, Chrysophyta, Charophyta, Xanthophyta, Dinoflagellata, and primitive Coelomata. We conclude that different proteins whose common feature is the presence of domains with a certain conformation are involved in the formation of the SC in different eukaryotic phyla. This permits a targeted search for orthologs of the SC proteins using phylogenetic trees. Here we consider example of phylogenetic trees for protozoans, fungi, algae, mosses, and flowering plants.

  9. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Zdenka Drastichova; Jiri Novotny

    2012-01-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors.At least some types of Gprotein o subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists.However,the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol.Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of Gq/1 1α protein and to determine their localization in isolated fractions and stability in naive and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G11α protein.We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the Gq/11 protein,which were found to be membrane-bound.Both of these complexes dissociated after prolonged treatment with TRH.Still other Gq/11α protein complexes of lower molecular weight were determined in the cytosol.These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h)hormone treatment.These results support the notion that a portion of Gq/11α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor.At the same time,these findings indicate that the redistribution of Gq/11α is broughtabout by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the Gq/11α protein.

  10. Correlation of mRNA and protein in complex biological samples.

    Science.gov (United States)

    Maier, Tobias; Güell, Marc; Serrano, Luis

    2009-12-17

    The correlation between mRNA and protein abundances in the cell has been reported to be notoriously poor. Recent technological advances in the quantitative analysis of mRNA and protein species in complex samples allow the detailed analysis of this pathway at the center of biological systems. We give an overview of available methods for the identification and quantification of free and ribosome-bound mRNA, protein abundances and individual protein turnover rates. We review available literature on the correlation of mRNA and protein abundances and discuss biological and technical parameters influencing the correlation of these central biological molecules.

  11. Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

    Science.gov (United States)

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-01-01

    The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  12. A Salmonella type three secretion effector/chaperone complex adopts a hexameric ring-like structure.

    Science.gov (United States)

    Roblin, Pierre; Dewitte, Frédérique; Villeret, Vincent; Biondi, Emanuele G; Bompard, Coralie

    2015-02-15

    Many bacterial pathogens use type three secretion systems (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS components are conserved among plant and animal pathogens, suggesting a common mechanism of recognition and secretion of effectors. However, no common motif has yet been identified for effectors allowing T3SS recognition. In this work, we performed a biochemical and structural characterization of the Salmonella SopB/SigE chaperone/effector complex by small-angle X-ray scattering (SAXS). Our results showed that the SopB/SigE complex is assembled in dynamic homohexameric-ring-shaped structures with an internal tunnel. In this ring, the chaperone maintains a disordered N-terminal end of SopB molecules, in a good position to be reached and processed by the T3SS. This ring dimensionally fits the ring-organized molecules of the injectisome, including ATPase hexameric rings; this organization suggests that this structural feature is important for ATPase recognition by T3SS. Our work constitutes the first evidence of the oligomerization of an effector, analogous to the organization of the secretion machinery, obtained in solution. As effectors share neither sequence nor structural identity, the quaternary oligomeric structure could constitute a strategy evolved to promote the specificity and efficiency of T3SS recognition.

  13. Application of model bread baking in the examination of arabinoxylan-protein complexes in rye bread.

    Science.gov (United States)

    Buksa, Krzysztof

    2016-09-01

    The changes in molecular mass of arabinoxylan (AX) and protein caused by bread baking process were examined using a model rye bread. Instead of the normal flour, the dough contained starch, water-extractable AX and protein which were isolated from rye wholemeal. From the crumb of selected model breads, starch was removed releasing AX-protein complexes, which were further examined by size exclusion chromatography. On the basis of the research, it was concluded that optimum model mix can be composed of 3-6% AX and 3-6% rye protein isolate at 94-88% of rye starch meaning with the most similar properties to low extraction rye flour. Application of model rye bread allowed to examine the interactions between AX and proteins. Bread baked with a share of AX, rye protein and starch, from which the complexes of the highest molar mass were isolated, was characterized by the strongest structure of the bread crumb. PMID:27185141

  14. Application of model bread baking in the examination of arabinoxylan-protein complexes in rye bread.

    Science.gov (United States)

    Buksa, Krzysztof

    2016-09-01

    The changes in molecular mass of arabinoxylan (AX) and protein caused by bread baking process were examined using a model rye bread. Instead of the normal flour, the dough contained starch, water-extractable AX and protein which were isolated from rye wholemeal. From the crumb of selected model breads, starch was removed releasing AX-protein complexes, which were further examined by size exclusion chromatography. On the basis of the research, it was concluded that optimum model mix can be composed of 3-6% AX and 3-6% rye protein isolate at 94-88% of rye starch meaning with the most similar properties to low extraction rye flour. Application of model rye bread allowed to examine the interactions between AX and proteins. Bread baked with a share of AX, rye protein and starch, from which the complexes of the highest molar mass were isolated, was characterized by the strongest structure of the bread crumb.

  15. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay

    Science.gov (United States)

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells.Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. Electronic supplementary information (ESI) available: Materials, experimental methods and Fig. S1-S8. See DOI: 10.1039/c6nr00171h

  16. Detection of Two Isomeric Binding Configurations in a Protein-Aptamer Complex with a Biological Nanopore

    NARCIS (Netherlands)

    Van Meervelt, Veerle; Soskine, Misha; Maglia, Giovanni

    2014-01-01

    ProteinDNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we showed that folded proteins can be internalized inside ClyA nanopores and studied by ionic current recordings a

  17. A human phenome-interactome network of protein complexes implicated in genetic disorders

    DEFF Research Database (Denmark)

    Hansen, Kasper Lage; Karlberg, Erik, Olof, Linnart; Størling, Zenia, Marian;

    2007-01-01

    We performed a systematic, large-scale analysis of human protein complexes comprising gene products implicated in many different categories of human disease to create a phenome-interactome network. This was done by integrating quality-controlled interactions of human proteins with a validated, co...

  18. Interaction of Small Zinc Complexes with Globular Proteins and Free Tryptophan

    Directory of Open Access Journals (Sweden)

    Joann M. Butkus

    2016-01-01

    Full Text Available A series of eight water soluble anionic, cationic, and neutral zinc(II complexes were synthesized and characterized. The interaction of these complexes with bovine serum albumin (BSA, human serum albumin (HSA, lysozyme, and free tryptophan (Trp was investigated using steady-state fluorescence spectroscopy. Static and dynamic fluorescence quenching analysis based on Stern-Volmer kinetics was conducted, and the decrease in fluorescence intensity of the Trp residue(s can be ascribed predominantly to static quenching that occurs when the Zn complex binds to the protein and forms a nonfluorescent complex. The role played by the nature of the ligand, the metal, and complex charge in quenching Trp fluorescence was investigated. The binding association constants (Ka ranged from 104 to 1010 M−1 and indicate that complexes with planar aromatic features have the strongest affinity for globular proteins and free Trp. Complexes with nonaromatic features failed to interact with these proteins at or in the vicinity of the Trp residues. These interactions were studied over a range of temperatures, and binding was found to weaken with the increase in temperature and was exothermic with a negative change in entropy. The thermodynamic parameters suggest that binding of Zn complexes to the proteins is a highly spontaneous and favorable process.

  19. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-26

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystallographic structures in the Protein Data Bank (PDB) by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of potential autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. We have identified 15 unique autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described in the relevant publications on the crystal structures (N-terminal juxtamembrane regions of CSF1R and EPHA2, activation loop tyrosines of LCK and IGF1R, and a serine in a nuclear localization signal region of CLK2. Mutation of residues in the autophosphorylation complex interface of LCK either severely impaired autophosphorylation or increased it. Taking the autophosphorylation complexes as a whole and comparing them with peptide-substrate/kinase complexes, we observe a number of important features among them. The novel and previously observed autophosphorylation sites are conserved in many kinases, indicating that by homology we can extend the relevance of these complexes to many other clinically relevant drug targets.

  20. A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes

    NARCIS (Netherlands)

    Patel, Roshani; Vasilev, Cvetelin; Beck, Daniel; Monteferrante, Carmine G; van Dijl, Jan Maarten; Hunter, C Neil; Smith, Corinne; Robinson, Colin

    2014-01-01

    The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report

  1. Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

    Directory of Open Access Journals (Sweden)

    Edyta Marcon

    2014-07-01

    Full Text Available Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  2. Physicochemical Characterization and Potential Prebiotic Effect of Whey Protein Isolate/Inulin Nano Complex

    OpenAIRE

    Ha, Ho-Kyung; Jeon, Na-Eun; Kim, Jin Wook; Han, Kyoung-Sik; Yun, Sung Seob; Lee, Mee-Ryung; Lee, Won-Jae

    2016-01-01

    The purposes of this study were to investigate the impacts of concentration levels of whey protein isolate (WPI) and inulin on the formation and physicochemical properties of WPI/inulin nano complexes and to evaluate their potential prebiotic effects. WPI/inulin nano complexes were produced using the internal gelation method. Transmission electron microscopy (TEM) and particle size analyzer were used to assess the morphological and physicochemical characterizations of nano complexes, respecti...

  3. Examination of tyrosine/adenine stacking interactions in protein complexes.

    Science.gov (United States)

    Copeland, Kari L; Pellock, Samuel J; Cox, James R; Cafiero, Mauricio L; Tschumper, Gregory S

    2013-11-14

    The π-stacking interactions between tyrosine amino acid side chains and adenine-bearing ligands are examined. Crystalline protein structures from the protein data bank (PDB) exhibiting face-to-face tyrosine/adenine arrangements were used to construct 20 unique 4-methylphenol/N9-methyladenine (p-cresol/9MeA) model systems. Full geometry optimization of the 20 crystal structures with the M06-2X density functional theory method identified 11 unique low-energy conformations. CCSD(T) complete basis set (CBS) limit interaction energies were estimated for all of the structures to determine the magnitude of the interaction between the two ring systems. CCSD(T) computations with double-ζ basis sets (e.g., 6-31G*(0.25) and aug-cc-pVDZ) indicate that the MP2 method overbinds by as much as 3.07 kcal mol(-1) for the crystal structures and 3.90 kcal mol(-1) for the optimized structures. In the 20 crystal structures, the estimated CCSD(T) CBS limit interaction energy ranges from -4.00 to -6.83 kcal mol(-1), with an average interaction energy of -5.47 kcal mol(-1), values remarkably similar to the corresponding data for phenylalanine/adenine stacking interactions. Geometry optimization significantly increases the interaction energies of the p-cresol/9MeA model systems. The average estimated CCSD(T) CBS limit interaction energy of the 11 optimized structures is 3.23 kcal mol(-1) larger than that for the 20 crystal structures.

  4. Iron-sulfur Proteins Are the Major Source of Protein-bound Dinitrosyl Iron Complexes Formed in Escherichia coli Cells under Nitric Oxide Stress

    OpenAIRE

    Landry, Aaron P.; Duan, Xuewu; Huang, Hao; Ding, Huangen

    2011-01-01

    Protein-bound dinitrosyl iron complexes (DNICs) have been observed in prokaryotic and eukaryotic cells under nitric oxide (NO) stress. The identity of proteins that bind DNICs, however, still remains elusive. Here we demonstrate that iron-sulfur proteins are the major source of protein-bound DNICs formed in Escherichia coli cells under NO stress. Expression of recombinant iron-sulfur proteins, but not the proteins without iron-sulfur clusters, almost doubles the amount of protein-bound DNICs ...

  5. Detection and identification of stable oligomeric protein complexes in Escherichi coli inner membranes: a proteomics approach.

    Science.gov (United States)

    Spelbrink, Robin E J; Kolkman, Annemieke; Slijper, Monique; Killian, J Antoinette; de Kruijff, Ben

    2005-08-01

    In this study we present a new technology to detect stable oligomeric protein complexes in membranes. The technology is based on the ability of small membrane-active alcohols to dissociate the highly stable homotetrameric potassium channel KcsA. It is shown via a proteomics approach, using diagonal electrophoresis and nano-flow liquid chromatography coupled to tandem mass spectrometry, that a large number of both integral and peripheral Escherichia coli inner membrane proteins are part of stable oligomeric complexes that can be dissociated by small alcohols. This study gives insight into the composition and stability of these complexes. PMID:15919657

  6. Analysis of Native-Like Proteins and Protein Complexes Using Cation to Anion Proton Transfer Reactions (CAPTR)

    Science.gov (United States)

    Laszlo, Kenneth J.; Bush, Matthew F.

    2015-12-01

    Mass spectra of native-like protein complexes often exhibit narrow charge-state distributions, broad peaks, and contributions from multiple, coexisting species. These factors can make it challenging to interpret those spectra, particularly for mixtures with significant heterogeneity. Here we demonstrate the use of ion/ion proton transfer reactions to reduce the charge states of m/ z-selected, native-like ions of proteins and protein complexes, a technique that we refer to as cation to anion proton transfer reactions (CAPTR). We then demonstrate that CAPTR can increase the accuracy of charge state assignments and the resolution of interfering species in native mass spectrometry. The CAPTR product ion spectra for pyruvate kinase exhibit ~30 peaks and enable unambiguous determination of the charge state of each peak, whereas the corresponding precursor spectra exhibit ~6 peaks and the assigned charge states have an uncertainty of ±3%. 15+ bovine serum albumin and 21+ yeast enolase dimer both appear near m/ z 4450 and are completely unresolved in a mixture. After a single CAPTR event, the resulting product ions are baseline resolved. The separation of the product ions increases dramatically after each subsequent CAPTR event; 12 events resulted in a 3000-fold improvement in separation relative to the precursor ions. Finally, we introduce a framework for interpreting and predicting the figures of merit for CAPTR experiments. More generally, these results suggest that CAPTR strongly complements other mass spectrometry tools for analyzing proteins and protein complexes, particularly those in mixtures.

  7. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  8. Node sampling for protein complex estimation in bait-prey graphs.

    Science.gov (United States)

    Scholtens, Denise M; Spencer, Bruce D

    2015-08-01

    In cellular biology, node-and-edge graph or "network" data collection often uses bait-prey technologies such as co-immunoprecipitation (CoIP). Bait-prey technologies assay relationships or "interactions" between protein pairs, with CoIP specifically measuring protein complex co-membership. Analyses of CoIP data frequently focus on estimating protein complex membership. Due to budgetary and other constraints, exhaustive assay of the entire network using CoIP is not always possible. We describe a stratified sampling scheme to select baits for CoIP experiments when protein complex estimation is the main goal. Expanding upon the classic framework in which nodes represent proteins and edges represent pairwise interactions, we define generalized nodes as sets of adjacent nodes with identical adjacency outside the set and use these as strata from which to select the next set of baits. Strata are redefined at each round of sampling to incorporate accumulating data. This scheme maintains user-specified quality thresholds for protein complex estimates and, relative to simple random sampling, leads to a marked increase in the number of correctly estimated complexes at each round of sampling. The R package seqSample contains all source code and is available at http://vault.northwestern.edu/~dms877/Rpacks/.

  9. Technical tip: high-resolution isolation of nanoparticle-protein corona complexes from physiological fluids

    Science.gov (United States)

    di Silvio, Desirè; Rigby, Neil; Bajka, Balazs; Mayes, Andrew; Mackie, Alan; Baldelli Bombelli, Francesca

    2015-07-01

    Nanoparticles (NPs) in contact with biological fluids are generally coated with environmental proteins, forming a stronger layer of proteins around the NP surface called the hard corona. Protein corona complexes provide the biological identity of the NPs and their isolation and characterization are essential to understand their in vitro and in vivo behaviour. Here we present a one-step methodology to recover NPs from complex biological media in a stable non-aggregated form without affecting the structure or composition of the corona. This method allows NPs to be separated from complex fluids containing biological particulates and in a form suitable for use in further experiments. The study has been performed systematically comparing the new proposed methodology to standard approaches for a wide panel of NPs. NPs were first incubated in the biological fluid and successively recovered by sucrose gradient ultracentrifugation in order to separate the NPs and their protein corona from the loosely bound proteins. The isolated NP-protein complexes were characterized by size and protein composition through Dynamic Light Scattering, Nanoparticle Tracking Analysis, SDS-PAGE and LC-MS. The protocol described is versatile and can be applied to diverse nanomaterials and complex fluids. It is shown to have higher resolution in separating the multiple protein corona complexes from a biological environment with a much lower impact on their in situ structure compared to conventional centrifugal approaches.Nanoparticles (NPs) in contact with biological fluids are generally coated with environmental proteins, forming a stronger layer of proteins around the NP surface called the hard corona. Protein corona complexes provide the biological identity of the NPs and their isolation and characterization are essential to understand their in vitro and in vivo behaviour. Here we present a one-step methodology to recover NPs from complex biological media in a stable non-aggregated form without

  10. On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes

    Science.gov (United States)

    Chen, Fan; Gerber, Sabina; Korkhov, Volodymyr M.; Mireku, Samantha; Bucher, Monika; Locher, Kaspar P.; Zenobi, Renato

    2015-03-01

    We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC2D2. We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.

  11. The role of protein complexes in a complex disease: molecular mechanisms of ALS

    NARCIS (Netherlands)

    Blokhuis, A.M.

    2016-01-01

    Amyotrophic Lateral Sclerosis is a devastating neurodegenerative diease caused by the selective loss of motor neurons. The pathogenic mechanism underlying the disease is largely unknown but a number of genes, proteins and cellular processes have been implicated. In this thesis we aimed to identify m

  12. Multilevel deconstruction of the In vivo behavior of looped DNA-protein complexes.

    Directory of Open Access Journals (Sweden)

    Leonor Saiz

    Full Text Available Protein-DNA complexes with loops play a fundamental role in a wide variety of cellular processes, ranging from the regulation of DNA transcription to telomere maintenance. As ubiquitous as they are, their precise in vivo properties and their integration into the cellular function still remain largely unexplored. Here, we present a multilevel approach that efficiently connects in both directions molecular properties with cell physiology and use it to characterize the molecular properties of the looped DNA-lac repressor complex while functioning in vivo. The properties we uncover include the presence of two representative conformations of the complex, the stabilization of one conformation by DNA architectural proteins, and precise values of the underlying twisting elastic constants and bending free energies. Incorporation of all this molecular information into gene-regulation models reveals an unprecedented versatility of looped DNA-protein complexes at shaping the properties of gene expression.

  13. The P-700-chlorophyl alpha-protein complex and two major light-harvesting complexes of Acrocarpia paniculata and other brown seaweeds.

    Science.gov (United States)

    Barrett, J; Anderson, J M

    1980-05-01

    Acrocarpia paniculata thylakoids were fragmented with Triton X-100 and the pigment-protein complexes so released were isolated by sucrose density gradient centrifugation. Three main chlorophyll-carotenoid-protein complexes with distinct pigment compositions were isolated. (1) A P-700-chlorophyll a-protein complex, with a ratio of 1 P-700: 38 chlorophyll a: 4 beta-carotene molecules, had similar absorption and fluorescence characteristics to the chlorophyll-protein complex 1 isolated with Triton X-100 from higher plants, green algae and Ecklonia radiata. (2) an orange-brown complex had a chlorophyll a : c2 : fucoxanthin molar ratio of 2 : 1 : 2. this complex had no chlorophyll c1 and contained most of the fucoxanthin present in the chloroplasts. This pigment complex is postulated to be the main light-harvesting complex of brown seaweeds. (3) A green complex had a chlorophyll a : c1 : c2 : violaxanthin molar ratio of 8 : 1 : 1. This also is a light-harvesting complex. the absorption and fluorescence spectral characteristics and other physical properties were consistent with the pigments of these three major complexes being bound to protein. Differential extraction of brown algal thylakoids with Triton X-100 showed that a chlorophyll c2-fucoxanthin-protein complex was a minor pigment complex of these thylakoids. PMID:7378391

  14. Interactions of the human MCM-BP protein with MCM complex components and Dbf4.

    Directory of Open Access Journals (Sweden)

    Tin Nguyen

    Full Text Available MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.

  15. Dynamics of nanoparticle-protein corona complex formation: analytical results from population balance equations.

    Directory of Open Access Journals (Sweden)

    Faryad Darabi Sahneh

    Full Text Available BACKGROUND: Nanoparticle-protein corona complex formation involves absorption of protein molecules onto nanoparticle surfaces in a physiological environment. Understanding the corona formation process is crucial in predicting nanoparticle behavior in biological systems, including applications of nanotoxicology and development of nano drug delivery platforms. METHOD: This paper extends the modeling work in to derive a mathematical model describing the dynamics of nanoparticle corona complex formation from population balance equations. We apply nonlinear dynamics techniques to derive analytical results for the composition of nanoparticle-protein corona complex, and validate our results through numerical simulations. RESULTS: The model presented in this paper exhibits two phases of corona complex dynamics. In the first phase, proteins rapidly bind to the free surface of nanoparticles, leading to a metastable composition. During the second phase, continuous association and dissociation of protein molecules with nanoparticles slowly changes the composition of the corona complex. Given sufficient time, composition of the corona complex reaches an equilibrium state of stable composition. We find analytical approximate formulae for metastable and stable compositions of corona complex. Our formulae are very well-structured to clearly identify important parameters determining corona composition. CONCLUSION: The dynamics of biocorona formation constitute vital aspect of interactions between nanoparticles and living organisms. Our results further understanding of these dynamics through quantitation of experimental conditions, modeling results for in vitro systems to better predict behavior for in vivo systems. One potential application would involve a single cell culture medium related to a complex protein medium, such as blood or tissue fluid.

  16. BiCAMWI: A Genetic-Based Biclustering Algorithm for Detecting Dynamic Protein Complexes.

    Science.gov (United States)

    Lakizadeh, Amir; Jalili, Saeed

    2016-01-01

    Considering the roles of protein complexes in many biological processes in the cell, detection of protein complexes from available protein-protein interaction (PPI) networks is a key challenge in the post genome era. Despite high dynamicity of cellular systems and dynamic interaction between proteins in a cell, most computational methods have focused on static networks which cannot represent the inherent dynamicity of protein interactions. Recently, some researchers try to exploit the dynamicity of PPI networks by constructing a set of dynamic PPI subnetworks correspondent to each time-point (column) in a gene expression data. However, many genes can participate in multiple biological processes and cellular processes are not necessarily related to every sample, but they might be relevant only for a subset of samples. So, it is more interesting to explore each subnetwork based on a subset of genes and conditions (i.e., biclusters) in a gene expression data. Here, we present a new method, called BiCAMWI to employ dynamicity in detecting protein complexes. The preprocessing phase of the proposed method is based on a novel genetic algorithm that extracts some sets of genes that are co-regulated under some conditions from input gene expression data. Each extracted gene set is called bicluster. In the detection phase of the proposed method, then, based on the biclusters, some dynamic PPI subnetworks are extracted from input static PPI network. Protein complexes are identified by applying a detection method on each dynamic PPI subnetwork and aggregating the results. Experimental results confirm that BiCAMWI effectively models the dynamicity inherent in static PPI networks and achieves significantly better results than state-of-the-art methods. So, we suggest BiCAMWI as a more reliable method for protein complex detection. PMID:27462706

  17. Synthetic strategies for efficient conjugation of organometallic complexes with pendant protein reactive markers

    KAUST Repository

    Jantke, Dominik

    2013-11-01

    Site-directed conjugation of metal centers to proteins is fundamental for biological and bioinorganic applications of transition metals. However, methods for the site-selective introduction of metal centers remain scarce. Herein, we present broadly applicable synthetic strategies for the conjugation of bioactive molecules with a range of organometallic complexes. Following three different synthetic strategies, we were able to synthesize a small library of metal conjugated protein markers featuring different types of protein reactive sites (epoxides, phenylphosphonates, fluorosulfonates and fluorophosphonate groups) as well as different late transition metals (iron, ruthenium, rhodium, palladium and platinum). The products were isolated in moderate to excellent yields and high purity. Furthermore, X-ray diffraction of the metalated protein markers corroborates structural integrity of the metal complex and the protein reactive site. © 2013 Elsevier B.V. All rights reserved.

  18. Proteomics analysis of Thermoplasma acidophilum with a focus on protein complexes.

    Science.gov (United States)

    Sun, Na; Beck, Florian; Knispel, Roland Wilhelm; Siedler, Frank; Scheffer, Beatrix; Nickell, Stephan; Baumeister, Wolfgang; Nagy, István

    2007-03-01

    Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.

  19. Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

    OpenAIRE

    Chu, Edward; Copur, Sitki M.; Ju, Jingfang; Chen, Tian-men; Khleif, Samir; Voeller, Donna M.; Mizunuma, Nobuyuki; Patel, Mahendra; Maley, Gladys F.; Maley, Frank; Allegra, Carmen J.

    1999-01-01

    A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRN...

  20. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    Energy Technology Data Exchange (ETDEWEB)

    Mena, Natalia P. [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Bulteau, Anne Laure [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Salazar, Julio [Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Hirsch, Etienne C. [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Nunez, Marco T., E-mail: mnunez@uchile.cl [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile)

    2011-06-03

    Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that

  1. ParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres.

    Science.gov (United States)

    Funnell, Barbara E

    2016-01-01

    In bacteria, active partition systems contribute to the faithful segregation of both chromosomes and low-copy-number plasmids. Each system depends on a site-specific DNA binding protein to recognize and assemble a partition complex at a centromere-like site, commonly called parS. Many plasmid, and all chromosomal centromere-binding proteins are dimeric helix-turn-helix DNA binding proteins, which are commonly named ParB. Although the overall sequence conservation among ParBs is not high, the proteins share similar domain and functional organization, and they assemble into similar higher-order complexes. In vivo, ParBs "spread," that is, DNA binding extends away from the parS site into the surrounding non-specific DNA, a feature that reflects higher-order complex assembly. ParBs bridge and pair DNA at parS and non-specific DNA sites. ParB dimers interact with each other via flexible conformations of an N-terminal region. This review will focus on the properties of the HTH centromere-binding protein, in light of recent experimental evidence and models that are adding to our understanding of how these proteins assemble into large and dynamic partition complexes at and around their specific DNA sites. PMID:27622187

  2. The Effect of Edge Definition of Complex Networks on Protein Structure Identification

    Directory of Open Access Journals (Sweden)

    Jing Sun

    2013-01-01

    Full Text Available The main objective of this study is to explore the contribution of complex network together with its different definitions of vertexes and edges to describe the structure of proteins. Protein folds into a specific conformation for its function depending on interactions between residues. Consequently, in many studies, a protein structure was treated as a complex system comprised of individual components residues, and edges were interactions between residues. What is the proper time for representing a protein structure as a network? To confirm the effect of different definitions of vertexes and edges in constructing the amino acid interaction networks, protein domains and the structural unit of proteins were described using this method. The identification performance of 2847 proteins with domain/domains proved that the structure of proteins was described well when was around 5.0–7.5 Å, and the optimal cutoff value for constructing the protein structure networks was 5.0 Å ( distances while the ideal community division method was community structure detection based on edge betweenness in this study.

  3. Computational analysis of interactions in structurally available protein-glycosaminoglycan complexes.

    Science.gov (United States)

    Samsonov, Sergey A; Pisabarro, M Teresa

    2016-08-01

    Glycosaminoglycans represent a class of linear anionic periodic polysaccharides, which play a key role in a variety of biological processes in the extracellular matrix via interactions with their protein targets. Computationally, glycosaminoglycans are very challenging due to their high flexibility, periodicity and electrostatics-driven nature of the interactions with their protein counterparts. In this work, we carry out a detailed computational characterization of the interactions in protein-glycosaminoglycan complexes from the Protein Data Bank (PDB), which are split into two subsets accounting for their intrinsic nature: non-enzymatic-protein-glycosaminoglycan and enzyme-glycosaminoglycan complexes. We apply molecular dynamics to analyze the differences in these two subsets in terms of flexibility, retainment of the native interactions in the simulations, free energy components of binding and contributions of protein residue types to glycosaminoglycan binding. Furthermore, we systematically demonstrate that protein electrostatic potential calculations, previously found to be successful for glycosaminoglycan binding sites prediction for individual systems, are in general very useful for proposing protein surface regions as putative glycosaminoglycan binding sites, which can be further used for local docking calculations with these particular polysaccharides. Finally, the performance of six different docking programs (Autodock 3, Autodock Vina, MOE, eHiTS, FlexX and Glide), some of which proved to perform well for particular protein-glycosaminoglycan complexes in previous work, is evaluated on the complete protein-glycosaminoglycan data set from the PDB. This work contributes to widen our knowledge of protein-glycosaminoglycan molecular recognition and could be useful to steer a choice of the strategies to be applied in theoretical studies of these systems. PMID:27496767

  4. Simplified Method for Predicting a Functional Class of Proteins in Transcription Factor Complexes

    KAUST Repository

    Piatek, Marek J.

    2013-07-12

    Background:Initiation of transcription is essential for most of the cellular responses to environmental conditions and for cell and tissue specificity. This process is regulated through numerous proteins, their ligands and mutual interactions, as well as interactions with DNA. The key such regulatory proteins are transcription factors (TFs) and transcription co-factors (TcoFs). TcoFs are important since they modulate the transcription initiation process through interaction with TFs. In eukaryotes, transcription requires that TFs form different protein complexes with various nuclear proteins. To better understand transcription regulation, it is important to know the functional class of proteins interacting with TFs during transcription initiation. Such information is not fully available, since not all proteins that act as TFs or TcoFs are yet annotated as such, due to generally partial functional annotation of proteins. In this study we have developed a method to predict, using only sequence composition of the interacting proteins, the functional class of human TF binding partners to be (i) TF, (ii) TcoF, or (iii) other nuclear protein. This allows for complementing the annotation of the currently known pool of nuclear proteins. Since only the knowledge of protein sequences is required in addition to protein interaction, the method should be easily applicable to many species.Results:Based on experimentally validated interactions between human TFs with different TFs, TcoFs and other nuclear proteins, our two classification systems (implemented as a web-based application) achieve high accuracies in distinguishing TFs and TcoFs from other nuclear proteins, and TFs from TcoFs respectively.Conclusion:As demonstrated, given the fact that two proteins are capable of forming direct physical interactions and using only information about their sequence composition, we have developed a completely new method for predicting a functional class of TF interacting protein partners

  5. Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex.

    Science.gov (United States)

    Savitsky, Mikhail; Kim, Maria; Kravchuk, Oksana; Schwartz, Yuri B

    2016-02-01

    Chromatin insulators are remarkable regulatory elements that can bring distant genomic sites together and block unscheduled enhancer-promoter communications. Insulators act via associated insulator proteins of two classes: sequence-specific DNA binding factors and "bridging" proteins. The latter are required to mediate interactions between distant insulator elements. Chromatin insulators are critical for correct expression of complex loci; however, their mode of action is poorly understood. Here, we use the Drosophila bithorax complex as a model to investigate the roles of the bridging proteins Cp190 and Mod(mdg4). The bithorax complex consists of three evolutionarily conserved homeotic genes Ubx, abd-A, and Abd-B, which specify anterior-posterior identity of the last thoracic and all abdominal segments of the fly. Looking at effects of CTCF, mod(mdg4), and Cp190 mutations on expression of the bithorax complex genes, we provide the first functional evidence that Mod(mdg4) acts in concert with the DNA binding insulator protein CTCF. We find that Mod(mdg4) and Cp190 are not redundant and may have distinct functional properties. We, for the first time, demonstrate that Cp190 is critical for correct regulation of the bithorax complex and show that Cp190 is required at an exceptionally strong Fub insulator to partition the bithorax complex into two topological domains.

  6. Compound complex enzymes and proteins of Stipa capillata from Semipalatinsk polygon

    International Nuclear Information System (INIS)

    The effects of low and high doses of irradiation near Semipalatinsk Atomic lake on the compound complex of different enzymes and proteins of leaves from different population of Stipa capillata are considered. 36 samples of Stipa capillata were analyzed by the iso-electrofocusing methods, native and SDS-electrophoresis. Levels of radioactivity effect on compound complex of peroxidase, esterase, acid phosphates and soluble proteins were found. SDS-PAGE and IEF methods did not show difference in peptides spectra between 36 populations of examined species. It means, that difference between contaminated and non-contaminated populations not so big as was expected. Compound complex soluble protein of Stipa capillata leaves changes under chronic doses of radioactivity. The difference in spectra between control and contaminated leaves make up 3-6 bands. Control leaves have more high molecular weight proteins than contaminated ones. Appearance of new bands is one of ways of plant adaptation. New components of enzymes spectra and soluble proteins were found. It was suggested, that gene mutation or post-translation modification of these proteins are result of chronic irradiation. To prove exactly genetic nature of this alteration aminoacids sequence for these proteins the DNA sequence of different Stipa capillata populations genomes were compared

  7. Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chengliang [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Zhang, Qiongdi [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Hang, Tianrong [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Tao, Yue [Shanghai Children’s Medical Center, 1678 Dongfang Road, Pudong, Shanghai 200120, People’s Republic of (China); Ma, Xukai [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Wu, Minhao; Zhang, Xuan, E-mail: xuanzbin@ustc.edu.cn; Zang, Jianye, E-mail: xuanzbin@ustc.edu.cn [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China)

    2015-08-28

    The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

  8. The Histone Deacetylase Complex 1 Protein of Arabidopsis Has the Capacity to Interact with Multiple Proteins Including Histone 3-Binding Proteins and Histone 1 Variants.

    Science.gov (United States)

    Perrella, Giorgio; Carr, Craig; Asensi-Fabado, Maria A; Donald, Naomi A; Páldi, Katalin; Hannah, Matthew A; Amtmann, Anna

    2016-05-01

    Intrinsically disordered proteins can adopt multiple conformations, thereby enabling interaction with a wide variety of partners. They often serve as hubs in protein interaction networks. We have previously shown that the Histone Deacetylase Complex 1 (HDC1) protein from Arabidopsis (Arabidopsis thaliana) interacts with histone deacetylases and quantitatively determines histone acetylation levels, transcriptional activity, and several phenotypes, including abscisic acid sensitivity during germination, vegetative growth rate, and flowering time. HDC1-type proteins are ubiquitous in plants, but they contain no known structural or functional domains. Here, we explored the protein interaction spectrum of HDC1 using a quantitative bimolecular fluorescence complementation assay in tobacco (Nicotiana benthamiana) epidermal cells. In addition to binding histone deacetylases, HDC1 directly interacted with histone H3-binding proteins and corepressor-associated proteins but not with H3 or the corepressors themselves. Surprisingly, HDC1 also was able to interact with variants of the linker histone H1. Truncation of HDC1 to the ancestral core sequence narrowed the spectrum of interactions and of phenotypic outputs but maintained binding to a H3-binding protein and to H1. Thus, HDC1 provides a potential link between H1 and histone-modifying complexes.

  9. Accurate refinement of docked protein complexes using evolutionary information and deep learning.

    Science.gov (United States)

    Akbal-Delibas, Bahar; Farhoodi, Roshanak; Pomplun, Marc; Haspel, Nurit

    2016-06-01

    One of the major challenges for protein docking methods is to accurately discriminate native-like structures from false positives. Docking methods are often inaccurate and the results have to be refined and re-ranked to obtain native-like complexes and remove outliers. In a previous work, we introduced AccuRefiner, a machine learning based tool for refining protein-protein complexes. Given a docked complex, the refinement tool produces a small set of refined versions of the input complex, with lower root-mean-square-deviation (RMSD) of atomic positions with respect to the native structure. The method employs a unique ranking tool that accurately predicts the RMSD of docked complexes with respect to the native structure. In this work, we use a deep learning network with a similar set of features and five layers. We show that a properly trained deep learning network can accurately predict the RMSD of a docked complex with 1.40 Å error margin on average, by approximating the complex relationship between a wide set of scoring function terms and the RMSD of a docked structure. The network was trained on 35000 unbound docking complexes generated by RosettaDock. We tested our method on 25 different putative docked complexes produced also by RosettaDock for five proteins that were not included in the training data. The results demonstrate that the high accuracy of the ranking tool enables AccuRefiner to consistently choose the refinement candidates with lower RMSD values compared to the coarsely docked input structures.

  10. Accurate refinement of docked protein complexes using evolutionary information and deep learning.

    Science.gov (United States)

    Akbal-Delibas, Bahar; Farhoodi, Roshanak; Pomplun, Marc; Haspel, Nurit

    2016-06-01

    One of the major challenges for protein docking methods is to accurately discriminate native-like structures from false positives. Docking methods are often inaccurate and the results have to be refined and re-ranked to obtain native-like complexes and remove outliers. In a previous work, we introduced AccuRefiner, a machine learning based tool for refining protein-protein complexes. Given a docked complex, the refinement tool produces a small set of refined versions of the input complex, with lower root-mean-square-deviation (RMSD) of atomic positions with respect to the native structure. The method employs a unique ranking tool that accurately predicts the RMSD of docked complexes with respect to the native structure. In this work, we use a deep learning network with a similar set of features and five layers. We show that a properly trained deep learning network can accurately predict the RMSD of a docked complex with 1.40 Å error margin on average, by approximating the complex relationship between a wide set of scoring function terms and the RMSD of a docked structure. The network was trained on 35000 unbound docking complexes generated by RosettaDock. We tested our method on 25 different putative docked complexes produced also by RosettaDock for five proteins that were not included in the training data. The results demonstrate that the high accuracy of the ranking tool enables AccuRefiner to consistently choose the refinement candidates with lower RMSD values compared to the coarsely docked input structures. PMID:26846813

  11. Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Jørgensen, Thomas J. D.; Koefoed, Klaus;

    2013-01-01

    in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads...... prior to the HDX-MS experiment. However, when studying protein complexes of more than two proteins, immobilization can possibly introduce steric limitations to the interactions. Here, we present a method based on the high affinity biotin-streptavidin interaction that allows selective capture...... of biotinylated proteins even under the extreme conditions for hydrogen/deuterium exchange quenching i.e. pH 2.5 and 0 °C. This biotin-streptavidin capture strategy allows hydrogen/deuterium exchange to occur in proteins in solution and enables characterization of specific proteins in heteromultimeric protein...

  12. pipsqueak Encodes a Factor Essential for Sequence-Specific Targeting of a Polycomb Group Protein Complex

    Institute of Scientific and Technical Information of China (English)

    Der-HwaHuang; Yuh-LongChang; Chih-ChaoYang; I-ChingPan; BalasKing

    2005-01-01

    The Polycomb (Pc) group (Pc-G) of repressors is essential for transcriptional silencing of homeotic genes that determine the axial development of metazoan animals. It is generally believed that the multimeric complexes formed by these proteins nucleate certain chromatin structures to silence promoter activity upon binding to Pc-G response elements (PRE). Little is known, however, about the molecular mechanism involved in sequence-specific binding of these complexes. Here, we show that an immunoa ffinity-purified Pc protein complex contains a DNA binding activity specific to the (GA), motif in a PRE from the bithoraxoid region. We found that this activity can be attributed primarily to the large protein isoform encoded by pipsqueak (psq) instead of to the well-characterized GAGA factor. The functional relevance ofpsq to the silencing mechanismis strongly supported by its synergistic interactions with a subset of Pc-G that cause misexpression of homeotic genes.

  13. Multiple Plasmodium falciparum Merozoite Surface Protein 1 Complexes Mediate Merozoite Binding to Human Erythrocytes.

    Science.gov (United States)

    Lin, Clara S; Uboldi, Alessandro D; Epp, Christian; Bujard, Hermann; Tsuboi, Takafumi; Czabotar, Peter E; Cowman, Alan F

    2016-04-01

    Successful invasion of human erythrocytes byPlasmodium falciparummerozoites is required for infection of the host and parasite survival. The early stages of invasion are mediated via merozoite surface proteins that interact with human erythrocytes. The nature of these interactions are currently not well understood, but it is known that merozoite surface protein 1 (MSP1) is critical for successful erythrocyte invasion. Here we show that the peripheral merozoite surface proteins MSP3, MSP6, MSPDBL1, MSPDBL2, and MSP7 bind directly to MSP1, but independently of each other, to form multiple forms of the MSP1 complex on the parasite surface. These complexes have overlapping functions that interact directly with human erythrocytes. We also show that targeting the p83 fragment of MSP1 using inhibitory antibodies inhibits all forms of MSP1 complexes and disrupts parasite growthin vitro.

  14. PROTEIN SEPARATION USING ULTRAFILTRATION - AN EXAMPLE OF MULTI-SCALE COMPLEX SYSTEMS

    Institute of Scientific and Technical Information of China (English)

    Zhanfeng Cui

    2005-01-01

    In this mini-review, the complexity of protein fractionation using ultrafiltration is discussed. The coupling of the system hydrodynamics, boundary layer transport, membrane permeation, electrostatic and hydrophobic interactions and its effects on protein transmission and membrane selectivity are analysed. Although ultrafiltration is promising for larger scale protein purification and also with outstanding advantages both technically and economically, much needs to be done to derive the.general guidance for membrane selection, process design and system operation. With fine tuning of operational and physiochemical conditions, the process can be greatly improved in terms of process productivity and protein purity. A coupled multi-scale approach might provide a way forward to analyse this complex system and improve the confidence in applying such a promising technology and predictability of the outcome.

  15. Transition metal complexes as mediator-titrants in protein redox potentiometry.

    Science.gov (United States)

    Bernhardt, Paul V; Chen, Kuan-I; Sharpe, Philip C

    2006-10-01

    A selection of nine macrocyclic Fe(III/II) and Co(III/II) transition metal complexes has been chosen to serve as a universal set of mediator-titrants in redox potentiometry of protein samples. The potential range spanned by these mediators is approximately from +300 to -700 mV vs the normal hydrogen electrode, which covers the range of most protein redox potentials accessible in aqueous solution. The complexes employed exhibit stability in both their oxidized and their reduced forms as well as pH-independent redox potentials within the range 6 < pH < 9. The mediators were also chosen on the basis of their very weak visible absorption maxima in both oxidation states, which will enable (for the first time) optical redox potentiometric titrations of proteins with relatively low extinction coefficients. This has previously been impractical with organic mediators, such as indoles, viologens and quinones, whose optical spectra interfere strongly with those of the protein.

  16. Spatiotemporal dynamics of early DNA damage response proteins on complex DNA lesions.

    Directory of Open Access Journals (Sweden)

    Frank Tobias

    Full Text Available The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP. Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on

  17. Protein complex formation and intranuclear dynamics of NAC1 in cancer cells.

    Science.gov (United States)

    Nakayama, Naomi; Kato, Hiroaki; Sakashita, Gyosuke; Nariai, Yuko; Nakayama, Kentaro; Kyo, Satoru; Urano, Takeshi

    2016-09-15

    Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300-500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells.

  18. Protein complex formation and intranuclear dynamics of NAC1 in cancer cells.

    Science.gov (United States)

    Nakayama, Naomi; Kato, Hiroaki; Sakashita, Gyosuke; Nariai, Yuko; Nakayama, Kentaro; Kyo, Satoru; Urano, Takeshi

    2016-09-15

    Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300-500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells. PMID:27424155

  19. Detecting coordinated regulation of multi-protein complexes using logic analysis of gene expression

    Directory of Open Access Journals (Sweden)

    Yeates Todd O

    2009-12-01

    Full Text Available Abstract Background Many of the functional units in cells are multi-protein complexes such as RNA polymerase, the ribosome, and the proteasome. For such units to work together, one might expect a high level of regulation to enable co-appearance or repression of sets of complexes at the required time. However, this type of coordinated regulation between whole complexes is difficult to detect by existing methods for analyzing mRNA co-expression. We propose a new methodology that is able to detect such higher order relationships. Results We detect coordinated regulation of multiple protein complexes using logic analysis of gene expression data. Specifically, we identify gene triplets composed of genes whose expression profiles are found to be related by various types of logic functions. In order to focus on complexes, we associate the members of a gene triplet with the distinct protein complexes to which they belong. In this way, we identify complexes related by specific kinds of regulatory relationships. For example, we may find that the transcription of complex C is increased only if the transcription of both complex A AND complex B is repressed. We identify hundreds of examples of coordinated regulation among complexes under various stress conditions. Many of these examples involve the ribosome. Some of our examples have been previously identified in the literature, while others are novel. One notable example is the relationship between the transcription of the ribosome, RNA polymerase and mannosyltransferase II, which is involved in N-linked glycan processing in the Golgi. Conclusions The analysis proposed here focuses on relationships among triplets of genes that are not evident when genes are examined in a pairwise fashion as in typical clustering methods. By grouping gene triplets, we are able to decipher coordinated regulation among sets of three complexes. Moreover, using all triplets that involve coordinated regulation with the ribosome

  20. Description and control of dissociation channels in gas-phase protein complexes

    Science.gov (United States)

    Thachuk, Mark; Fegan, Sarah K.; Raheem, Nigare

    2016-08-01

    Using molecular dynamics simulations of a coarse-grained model of the charged apo-hemoglobin protein complex, this work expands upon our initial report [S. K. Fegan and M. Thachuk, J. Am. Soc. Mass Spectrom. 25, 722-728 (2014)] about control of dissociation channels in the gas phase using specially designed charge tags. Employing a charge hopping algorithm and a range of temperatures, a variety of dissociation channels are found for activated gas-phase protein complexes. At low temperatures, a single monomer unfolds and becomes charge enriched. At higher temperatures, two additional channels open: (i) two monomers unfold and charge enrich and (ii) two monomers compete for unfolding with one eventually dominating and the other reattaching to the complex. At even higher temperatures, other more complex dissociation channels open with three or more monomers competing for unfolding. A model charge tag with five sites is specially designed to either attract or exclude charges. By attaching this tag to the N-terminus of specific monomers, the unfolding of those monomers can be decidedly enhanced or suppressed. In other words, using charge tags to direct the motion of charges in a protein complex provides a mechanism for controlling dissociation. This technique could be used in mass spectrometry experiments to direct forces at specific attachment points in a protein complex, and hence increase the diversity of product channels available for quantitative analysis. In turn, this could provide insight into the function of the protein complex in its native biological environment. From a dynamics perspective, this system provides an interesting example of cooperative behaviour involving motions with differing time scales.

  1. Counterions release from electrostatic complexes of polyelectrolytes and proteins of opposite charge : a direct measurement

    CERN Document Server

    Gummel, Jérémie; Boué, François

    2009-01-01

    Though often considered as one of the main driving process of the complexation of species of opposite charges, the release of counterions has never been experimentally directly measured on polyelectrolyte/proteins complexes. We present here the first structural determination of such a release by Small Angle Neutron Scattering in complexes made of lysozyme, a positively charged protein and of PSS, a negatively charged polyelectrolyte. Both components have the same neutron density length, so their scattering can be switched off simultaneously in an appropriate "matching" solvent; this enables determination of the spatial distribution of the single counterions within the complexes. The counterions (including the one subjected to Manning condensation) are expelled from the cores where the species are at electrostatic stoichiometry.

  2. A Polycomb complex remains bound through DNA replication in the absence of other eukaryotic proteins

    KAUST Repository

    Lengsfeld, Bettina M.

    2012-09-17

    Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.

  3. Targeting a Dynamic Protein–Protein Interaction: Fragment Screening against the Malaria Myosin A Motor Complex

    OpenAIRE

    Douse, Christopher H; Vrielink, Nina; Wenlin, Zhang; Cota, Ernesto; Tate, Edward W

    2014-01-01

    Motility is a vital feature of the complex life cycle of Plasmodium falciparum, the apicomplexan parasite that causes human malaria. Processes such as host cell invasion are thought to be powered by a conserved actomyosin motor (containing myosin A or myoA), correct localization of which is dependent on a tight interaction with myosin A tail domain interacting protein (MTIP) at the inner membrane of the parasite. Although disruption of this protein–protein interaction represents an attractive...

  4. Fast automated placement of polar hydrogen atoms in protein-ligand complexes

    OpenAIRE

    Lippert Tobias; Rarey Matthias

    2009-01-01

    Abstract Background Hydrogen bonds play a major role in the stabilization of protein-ligand complexes. The ability of a functional group to form them depends on the position of its hydrogen atoms. An accurate knowledge of the positions of hydrogen atoms in proteins is therefore important to correctly identify hydrogen bonds and their properties. The high mobility of hydrogen atoms introduces several degrees of freedom: Tautomeric states, where a hydrogen atom alters its binding partner, torsi...

  5. 3D pressure field in lipid membranes and membrane-protein complexes

    DEFF Research Database (Denmark)

    Ollila, O H Samuli; Risselada, H Jelger; Louhivuori, Martti;

    2009-01-01

    a protein-membrane complex. We show that the 3D pressure field is distinctly different for curved and planar bilayers, the pressure field depends strongly on the phase of the membrane, and that an integral protein modulates the tension and elastic properties of the membrane.......We calculate full 3D pressure fields for inhomogeneous nanoscale systems using molecular dynamics simulation data. The fields represent systems with increasing level of complexity, ranging from semivesicles and vesicles to membranes characterized by coexistence of two phases, including also...

  6. Discovering New Features of Protein Complexes Structures by Small-Angle X-Ray Scattering

    Science.gov (United States)

    Oliveira, C. L. P.; Vorup-Jensen, T.; Andersen, C. B. F.; Andersen, G. R.; Pedersen, J. S.

    In spite of the recent advances in the X-Ray crystallography and nuclear magnetic resonance techniques, the determination of the quaternary structure of large protein complexes is still a challenge in molecular biology and biological sciences. In this respect, small-angle X-ray scattering (SAXS) is a key technique, enabling the determination of the possible structural conformation of complexes in an almost native state. Despite of this book being devoted to scattering techniques by synchrotron radiation, in this chapter we present two examples of application of laboratory-based SAXS to protein solution. The fundaments of the technique are obviously the same and have been deeply described in Chap. 2. In this chapter, we will introduce the application of SAXS to protein solution. Special emphasis is done on data reduction and absolute units calibration. As an example to illustrate the power of this technique, two new data sets for two protein complexes will be presented. This will show how high-quality SAXS data combined with advanced model strategies enables the determination of the quaternary structure of protein complexes.

  7. Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA

    Directory of Open Access Journals (Sweden)

    Celma Cristina C

    2009-09-01

    Full Text Available Abstract Background Many cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way. Results This paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins. Conclusion 1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in Ac

  8. Events during Initiation of Archaeal Transcription: Open Complex Formation and DNA-Protein Interactions

    OpenAIRE

    Hausner, Winfried; Thomm, Michael

    2001-01-01

    Transcription in Archaea is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon Methanococcus, in contrast to polymerase II, does not require hydrolysis of the β-γ bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex s...

  9. Finite size and inner structure controlled by electrostatic screening in globular complexes of proteins and polyelectrolytes

    OpenAIRE

    Gummel, Jérémie; Boué, François; Clemens, Daniel; Cousin, Fabrice

    2010-01-01

    International audience We present an extended structural study of globular complexes made by mixing a positively charge protein (lysozyme) and a negatively charged polyelectrolyte (PSS). We study the influence of all the parameters that may act on the structure of the complexes (charge densities and concentration of the species, partial hydrophobicity of the polyion chain, ionic strength). The structures on a 15 scale range lying from 10Å to 1000Å are measured by SANS. Whatever the conditi...

  10. Insulin/poly(ethylene glycol)-block-poly(L-lysine) Complexes: Physicochemical Properties and Protein Encapsulation.

    Science.gov (United States)

    Pippa, Natassa; Kalinova, Radostina; Dimitrov, Ivaylo; Pispas, Stergios; Demetzos, Costas

    2015-06-01

    Insulin (INS) was encapsulated into complexes with poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLys), which is a polypeptide-based block copolymer (a neutral-cationic block polyelectrolyte). The particular cationic-neutral block copolymer can complex INS molecules in aqueous media via electrostatic interactions. Light-scattering techniques are used to study the complexation process and structure of the hybrid nanoparticles in a series of buffers, as a function of protein concentration. The physicochemical and structural characteristics of the complexes depend on the ionic strength of the aqueous medium, while the concentration of PEG-b-PLys was constant through the series of solutions. As INS concentration increased the size distribution of the complexes decreased, especially at the highest ionic strength. The size/structure of complexes diluted in biological medium indicated that the copolymer imparts stealth properties and colloidal and biological stability to the complexes, features that could in turn affect the clearance properties in vivo. Therefore, these studies could be a rational roadmap for designing the optimum complexes/effective nanocarriers for proteins and peptides. PMID:25974620

  11. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    Science.gov (United States)

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-02-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex. PMID:3100577

  12. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    Science.gov (United States)

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  13. Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex.

    Science.gov (United States)

    Peña, A; Reddy, C D; Wu, S; Hickok, N J; Reddy, E P; Yumet, G; Soprano, D R; Soprano, K J

    1993-12-25

    The presence of a CACGTG element within a region of the human ornithine decarboxylase (ODC) promoter located at -491 to -474 base pairs 5' to the start site of transcription suggested that the c-Myc.Max protein complex may play a role in the regulation of ODC expression during growth. Electrophoretic mobility shift assays and methylation interference analysis showed that the nuclei of WI-38 cells expressing ODC contained proteins that bound to this region of the ODC gene in a manner that correlated with growth-associated ODC expression. Also, use of antibodies against c-Myc and Max and purified recombinant c-Myc and Max protein in the electrophoretic mobility shift assay confirmed that these proteins can specifically bind this portion of the human ODC promoter. Transient transfection studies showed that increase in the level of c-Myc and/or Max led to a significant enhancement of expression of a human ODC promoter-CAT reporter construct. Moreover, treatment of actively growing WI-38 cells with an antisense oligomer to c-Myc reduced the amount of endogenous protein complex formed and the amount of endogenous ODC mRNA expressed. These studies show that the c-Myc.Max protein complex plays a role in the transcriptional regulation of human ODC in vivo.

  14. Complex Assembly Behavior During the Encapsulation of Green Fluorescent Protein Analogs in Virus Derived Protein Capsules

    NARCIS (Netherlands)

    Minten, Inge J.; Nolte, Roeland J.M.; Cornelissen, Jeroen J.L.M.

    2010-01-01

    Enzymes encapsulated in nanocontainers are a better model of the conditions inside a living cell than free enzymes in solution. In a first step toward the encapsulation of multiple enzymes inside the cowpea chlorotic mottle virus (CCMV) capsid, enhanced green fluorescent protein (EGFP) was attached

  15. Taxonomic distribution and origins of the extended LHC (light-harvesting complex antenna protein superfamily

    Directory of Open Access Journals (Sweden)

    Brinkmann Henner

    2010-07-01

    Full Text Available Abstract Background The extended light-harvesting complex (LHC protein superfamily is a centerpiece of eukaryotic photosynthesis, comprising the LHC family and several families involved in photoprotection, like the LHC-like and the photosystem II subunit S (PSBS. The evolution of this complex superfamily has long remained elusive, partially due to previously missing families. Results In this study we present a meticulous search for LHC-like sequences in public genome and expressed sequence tag databases covering twelve representative photosynthetic eukaryotes from the three primary lineages of plants (Plantae: glaucophytes, red algae and green plants (Viridiplantae. By introducing a coherent classification of the different protein families based on both, hidden Markov model analyses and structural predictions, numerous new LHC-like sequences were identified and several new families were described, including the red lineage chlorophyll a/b-binding-like protein (RedCAP family from red algae and diatoms. The test of alternative topologies of sequences of the highly conserved chlorophyll-binding core structure of LHC and PSBS proteins significantly supports the independent origins of LHC and PSBS families via two unrelated internal gene duplication events. This result was confirmed by the application of cluster likelihood mapping. Conclusions The independent evolution of LHC and PSBS families is supported by strong phylogenetic evidence. In addition, a possible origin of LHC and PSBS families from different homologous members of the stress-enhanced protein subfamily, a diverse and anciently paralogous group of two-helix proteins, seems likely. The new hypothesis for the evolution of the extended LHC protein superfamily proposed here is in agreement with the character evolution analysis that incorporates the distribution of families and subfamilies across taxonomic lineages. Intriguingly, stress-enhanced proteins, which are universally found in the

  16. MPP1 links the Usher protein network and the Crumbs protein complex in the retina.

    NARCIS (Netherlands)

    Gosens, I.; Wijk, E. van; Kersten, F.F.J.; Krieger, E.; Zwaag, B. van der; Marker, T.; Letteboer, S.J.F.; Dusseljee, S.; Peters, T.; Spierenburg, H.A.; Punte, I.M.; Wolfrum, U.; Cremers, F.P.M.; Kremer, H.; Roepman, R.

    2007-01-01

    The highly ordered distribution of neurons is an essential feature of a functional mammalian retina. Disruptions in the apico-basal polarity complexes at the outer limiting membrane (OLM) of the retina are associated with retinal patterning defects in vertebrates. We have analyzed the binding repert

  17. The RNA Polymerase II CTD: The Increasing Complexity of a Low-Complexity Protein Domain.

    Science.gov (United States)

    Jeronimo, Célia; Collin, Pierre; Robert, François

    2016-06-19

    The largest subunit of RNA polymerase II contains a C-terminal domain (CTD) that plays key roles in coordinating transcription with co-transcriptional events. The heptapeptide repeats that form the CTD are dynamically phosphorylated on serine, tyrosine and threonine residues during the various steps of transcription, thereby regulating the recruitment of various proteins involved in gene expression. In this "Perspective," we review the recent literature related to the function of the CTD, to CTD kinases (Kin28, CDK7, CDK9, CDK12, ERK1/2 and DYRK1A) and to CTD phosphatases (Rtr1, RPAP2, Ssu72, Fcp1 and Gcl7). We discuss unresolved and controversial issues and try to provide constructive suggestions. This review also highlights emerging themes in the CTD field, such as crosstalk and feedback mechanisms, as well as gene-specific and tissue-specific functions of the CTD. Finally, promising therapeutic avenues for a recently developed CTD kinase inhibitor are discussed. PMID:26876604

  18. Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.

    Science.gov (United States)

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A; Lowe, Edward D; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W; Cogdell, Richard J; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-08-28

    How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups.

  19. Protein-Protein Interactions between Intermediate Chains and the Docking Complex of Chlamydomonas Flagellar Outer Arm Dynein

    Science.gov (United States)

    Ide, Takahiro; Owa, Mikito; King, Stephen M.; Kamiya, Ritsu; Wakabayashi, Ken-ichi

    2013-01-01

    Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association. PMID:23747306

  20. Physicochemical Characterization and Potential Prebiotic Effect of Whey Protein Isolate/Inulin Nano Complex.

    Science.gov (United States)

    Ha, Ho-Kyung; Jeon, Na-Eun; Kim, Jin Wook; Han, Kyoung-Sik; Yun, Sung Seob; Lee, Mee-Ryung; Lee, Won-Jae

    2016-01-01

    The purposes of this study were to investigate the impacts of concentration levels of whey protein isolate (WPI) and inulin on the formation and physicochemical properties of WPI/inulin nano complexes and to evaluate their potential prebiotic effects. WPI/inulin nano complexes were produced using the internal gelation method. Transmission electron microscopy (TEM) and particle size analyzer were used to assess the morphological and physicochemical characterizations of nano complexes, respectively. The encapsulation efficiency of resveratrol in nano complexes was studied using HPLC while the potential prebiotic effects were investigated by measuring the viability of probiotics. In TEM micrographs, the globular forms of nano complexes in the range of 10 and 100 nm were successfully manufactured. An increase in WPI concentration level from 1 to 3% (w/v) resulted in a significant (pnano complexs while inulin concentration level did not affect the size of nano complexes. The polydispersity index of nano complexes was below 0.3 in all cases while the zeta-potential values in the range of -2 and -12 mV were observed. The encapsulation efficiency of resveratrol was significantly (pnano complexes exhibited similar viability of probiotics with free inulin and had significantly (pnano complexes and had potential prebiotic effect.

  1. Complex viscosity induced by protein composition variation influences the aroma release of flavored stirred yogurt.

    Science.gov (United States)

    Saint-Eve, Anne; Juteau, Alexandre; Atlan, Samuel; Martin, Nathalie; Souchon, Isabelle

    2006-05-31

    Dairy protein composition is known to influence the structure and the texture characteristics of yogurt. The objective of the present work was therefore to investigate the impact of protein composition, at a constant protein level, on the physicochemical properties of 4% fat flavored stirred yogurt and, more specifically, on the rheological properties, the microstructure, and the aroma release. The results showed that caseinate-enriched yogurt generally presented changes in their microstructure network and had a higher complex viscosity than whey protein-enriched yogurt. To a lesser extent, the release of the majority of aroma compounds was lower in caseinate-enriched yogurt. It was therefore possible to quantify physicochemical interactions between aroma compounds and proteins. The influence of gel structure on the flavor release was observed and was in agreement with sensory characteristics previously studied for these products.

  2. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

    Science.gov (United States)

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

    2016-01-01

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

  3. Amplified DNA Detection Sensitivity Using Streptavidin-Biotinylated Protein Complex: Characterization by Electrochemical Impedance Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    CHENG, Zhi-Liang; YANG, Fan; HUANG, Hai-Zhen; YANG, Xiu-Rong

    2003-01-01

    Thiol-terminated oligonucleotide was immobilized to gold surface by self-assembly method. A novel amplification strategy was introduced for improving the sensitivity of DNA hybridization using biotin labeled protein-streptavidin network complex.This complex can be formed in a cross-linking network of molecules so that the amplification of the response signal will be realized due to the big molecular size of the complex. It could be proved from the impedance technique that this amplification strategy caused dramatic improvement of the detection sensitivity. These results give significant advances in the generality and sensitivity as it is applied to biosensing.

  4. Lipid binding in rice nonspecific lipid transfer protein-1 complexes from Oryza sativa

    OpenAIRE

    Cheng, Hui-Chun; Cheng, Pei-Tsung; Peng, Peiyu; Lyu, Ping-Chiang; Sun, Yuh-Ju

    2004-01-01

    Nonspecific lipid transfer proteins (nsLTPs) facilitate the transfer of phospholipids, glycolipids, fatty acids and steroids between membranes, with wide-ranging binding affinities. Three crystal structures of rice nsLTP1 from Oryza sativa, complexed with myristic (MYR), palmitic (PAL) or stearic acid (STE) were determined. The overall structures of the rice nsLTP1 complexes belong to the four-helix bundle folding with a long C-terminal loop. The nsLTP1–MYR and the nsLTP1–STE complexes bind a...

  5. Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

    Energy Technology Data Exchange (ETDEWEB)

    Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta; Yu, Carol; Abraham, Thomas; Borchers, Christoph [UBC James Hogg Research Centre, Institute for Heart and Lung Health, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia (Canada); University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia (Canada); Bernatchez, Pascal, E-mail: pbernatc@mail.ubc.ca [UBC James Hogg Research Centre, Institute for Heart and Lung Health, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia (Canada); University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia (Canada)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. Black-Right-Pointing-Pointer Dysferlin interacts with key signaling proteins for transcytosis in EC. Black-Right-Pointing-Pointer Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.

  6. SAMPLEX: Automatic mapping of perturbed and unperturbed regions of proteins and complexes

    Directory of Open Access Journals (Sweden)

    Loth Karine

    2010-01-01

    Full Text Available Abstract Background The activity of proteins within the cell is characterized by their motions, flexibility, interactions or even the particularly intriguing case of partially unfolded states. In the last two cases, a part of the protein is affected either by binding or unfolding and the detection of the respective perturbed and unperturbed region(s is a fundamental part of the structural characterization of these states. This can be achieved by comparing experimental data of the same protein in two different states (bound/unbound, folded/unfolded. For instance, measurements of chemical shift perturbations (CSPs from NMR 1H-15N HSQC experiments gives an excellent opportunity to discriminate both moieties. Results We describe an innovative, automatic and unbiased method to distinguish perturbed and unperturbed regions in a protein existing in two distinct states (folded/partially unfolded, bound/unbound. The SAMPLEX program takes as input a set of data and the corresponding three-dimensional structure and returns the confidence for each residue to be in a perturbed or unperturbed state. Its performance is demonstrated for different applications including the prediction of disordered regions in partially unfolded proteins and of interacting regions in protein complexes. Conclusions The proposed approach is suitable for partially unfolded states of proteins, local perturbations due to small ligands and protein-protein interfaces. The method is not restricted to NMR data, but is generic and can be applied to a wide variety of information.

  7. Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

    International Nuclear Information System (INIS)

    Highlights: ► Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. ► Dysferlin interacts with key signaling proteins for transcytosis in EC. ► Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.

  8. Dynamic spatial organization of multi-protein complexes controlling microbial polar organization, chromosome replication, and cytokinesis

    Energy Technology Data Exchange (ETDEWEB)

    McAdams, Harley; Shapiro, Lucille; Horowitz, Mark; Andersen, Gary; Downing, Kenneth; Earnest, Thomas; Ellisman, Mark; Gitai, Zemer; Larabell, Carolyn; Viollier, Patrick

    2012-06-18

    This project was a program to develop high-throughput methods to identify and characterize spatially localized multiprotein complexes in bacterial cells. We applied a multidisciplinary systems engineering approach to the detailed characterization of localized multi-protein structures in vivo a problem that has previously been approached on a fragmented, piecemeal basis.

  9. Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene

    NARCIS (Netherlands)

    Brzozowska, Agata M.; de Keizer, Arie; Norde, Willem; Detrembleur, Christophe; Stuart, Martien Cohen

    2010-01-01

    We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins beta-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer

  10. Small-World Effect of Complex Network and Its Application toProtein Folding

    Institute of Scientific and Technical Information of China (English)

    卢全国; 陈宝方; 彭华魁; 祖巧红

    2004-01-01

    The famous "six letters" experiment carried out by Milgram demonstrated the existence of small-world effect in a complex network. One vertex tends to be connected to another by a shortest path through network because of the small-world effect. This paper uses the small-world effect to study protein folding pathway.

  11. Single particle electron microscopy in combination with mass spectrometry to investigate novel complexes of membrane proteins

    NARCIS (Netherlands)

    Arteni, Ana A.; Nowaczyk, Marc; Lax, Julia; Kouřil, Roman; Rögner, Matthias; Boekema, Egbert J.; Kouril, R.; Rogner, M.

    2005-01-01

    Large data sets of molecular projections of the membrane proteins Photosystem I and Photosystem II from cyanobacteria were analyzed by single particle electron microscopy (EM). Analysis resulted in the averaging of 2D projections from the purified complexes but also in the simultaneous detection and

  12. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua;

    2012-01-01

    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  13. Oxalic acid complexes: Promising draw solutes for forward osmosis (FO) in protein enrichment

    KAUST Repository

    Ge, Qingchun

    2015-01-01

    Highly soluble oxalic acid complexes (OACs) were synthesized through a one-pot reaction. The OACs exhibit excellent performance as draw solutes in FO processes with high water fluxes and negligible reverse solute fluxes. Efficient protein enrichment was achieved. The diluted OACs can be recycled via nanofiltration and are promising as draw solutes.

  14. SCP2, a major protein component of the axial elements of synaptonemal complexes

    NARCIS (Netherlands)

    Schalk, M.

    1999-01-01

    Synaptonemal complexes (SCs) are ladderlike protein structures, which are formed between homologous chromosomes during the prophase of the first meiotic division. SCs consist of two axial elements, one along each chromosome, and transverse filaments (TFs), which connect the axial elements. On the TF

  15. Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene

    NARCIS (Netherlands)

    Brzozowska, A.M.; Keizer, de A.; Norde, W.; Detrembleur, C.; Cohen Stuart, M.A.

    2010-01-01

    We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins ß-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer PA

  16. Grafted ionomer complexes and their effect on protein adsorption on silica and polysulfone surfaces

    NARCIS (Netherlands)

    Brzozowska, Agata M.; de Keizer, Arie; Detrembleur, Christophe; Stuart, Martien A. Cohen; Norde, Willem

    2010-01-01

    We have studied the formation and the stability of ionomer complexes from grafted copolymers (GICs) in solution and the influence of GIC coatings on the adsorption of the proteins beta-lactoglobulin (beta-lac), bovine serum albumin (BSA), and lysozyme (Lsz) on silica and polysulfone. The GICs consis

  17. Long Unfolded Linkers Facilitate Membrane Protein Import Through the Nuclear Pore Complex

    NARCIS (Netherlands)

    Meinema, Anne C.; Laba, Justyna K.; Hapsari, Rizqiya A.; Otten, Renee; Mulder, Frans A. A.; Kralt, Annemarie; van den Bogaart, Geert; Lusk, C. Patrick; Poolman, Bert; Veenhoff, Liesbeth M.

    2011-01-01

    Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a

  18. The transport of integral membrane proteins across the nuclear pore complex

    NARCIS (Netherlands)

    Meinema, Anne C.; Poolman, Bert; Veenhoff, Liesbeth M.

    2012-01-01

    The nuclear envelope protects and organizes the genome. The nuclear pore complexes embedded in the nuclear envelope allow selective transport of macromolecules between the cytosol and nucleoplasm, and as such help to control the flow of information from DNA to RNA to proteins. A growing list of inte

  19. Unraveling the Electronic Structure of Individual Photosynthetic Pigment-Protein Complexes

    NARCIS (Netherlands)

    Oijen, Antoine M. van; Ketelaars, Martijn; Köhler, Jürgen; Aartsma, Thijs J.; Schmidt, Jan

    1999-01-01

    Low-temperature single-molecule spectroscopic techniques were applied to a light-harvesting pigment-protein complex (LH2) from purple photosynthetic bacteria. The properties of the electronically excited states of the two circular assemblies (B800 and B850) of bacteriochlorophyll a (BChl a) pigment

  20. Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

    DEFF Research Database (Denmark)

    Jönsson, Peter; Southcombe, Jennifer H; Santos, Ana Mafalda;

    2016-01-01

    value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with...

  1. Role for a novel Usher protein complex in hair cell synaptic maturation.

    Directory of Open Access Journals (Sweden)

    Marisa Zallocchi

    Full Text Available The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23, protocadherin-15 (PCDH15 and the very large G-protein coupled receptor 1 (VLGR1 have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1-/- mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzer(av3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well.

  2. Preparation of the Human Cytomegalovirus Nuclear Egress Complex and Associated Proteins.

    Science.gov (United States)

    Sharma, Mayuri; Kamil, Jeremy P; Coen, Donald M

    2016-01-01

    Herpesviruses, like most DNA viruses, replicate their genomes in the host cell nucleus. Their DNA is then packaged and assembled into viral nucleocapsids, which, in most cases, are too large to pass through the nuclear pore complex. Instead, herpesviruses use a complex multistep pathway, termed nuclear egress, to exit the nucleus. Key players in this process include two conserved viral proteins that form the nuclear egress complex (NEC). In human cytomegalovirus, these NEC proteins are UL50, embedded in the inner nuclear membrane, and its nucleoplasmic partner UL53. Both are essential for viral nuclear egress. However, other viral components as well as host nuclear envelope proteins may also participate in nuclear egress. Identifying these viral and cellular factors may provide important insight into the herpesvirus lifecycle and its relationship to the underlying, yet still-mysterious, host nuclear egress pathway. We developed an immunoprecipitation-based protocol, described herein, to identify protein-protein interactions involving the NEC from the nuclear fraction of infected cells that express an epitope-tagged version of NEC subunit UL53.

  3. Active-state models of ternary GPCR complexes: determinants of selective receptor-G-protein coupling.

    Directory of Open Access Journals (Sweden)

    Ralf C Kling

    Full Text Available Based on the recently described crystal structure of the β2 adrenergic receptor--Gs-protein complex, we report the first molecular-dynamics simulations of ternary GPCR complexes designed to identify the selectivity determinants for receptor-G-protein binding. Long-term molecular dynamics simulations of agonist-bound β2AR-Gαs and D2R-Gαi complexes embedded in a hydrated bilayer environment and computational alanine-scanning mutagenesis identified distinct residues of the N-terminal region of intracellular loop 3 to be crucial for coupling selectivity. Within the G-protein, specific amino acids of the α5-helix, the C-terminus of the Gα-subunit and the regions around αN-β1 and α4-β6 were found to determine receptor recognition. Knowledge of these determinants of receptor-G-protein binding selectivity is essential for designing drugs that target specific receptor/G-protein combinations.

  4. Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins

    Directory of Open Access Journals (Sweden)

    Coulson Richard MR

    2007-02-01

    Full Text Available Abstract Background In membrane trafficking, the mechanisms ensuring vesicle fusion specificity remain to be fully elucidated. Early models proposed that specificity was encoded entirely by SNARE proteins; more recent models include contributions from Rab proteins, Syntaxin-binding (SM proteins and tethering factors. Most information on membrane trafficking derives from an evolutionarily narrow sampling of model organisms. However, considering factors from a wider diversity of eukaryotes can provide both functional information on core systems and insight into the evolutionary history of the trafficking machinery. For example, the major Qa/syntaxin SNARE families are present in most eukaryotic genomes and likely each evolved via gene duplication from a single ancestral syntaxin before the existing eukaryotic groups diversified. This pattern is also likely for Rabs and various other components of the membrane trafficking machinery. Results We performed comparative genomic and phylogenetic analyses, when relevant, on the SM proteins and components of the tethering complexes, both thought to contribute to vesicle fusion specificity. Despite evidence suggestive of secondary losses amongst many lineages, the tethering complexes are well represented across the eukaryotes, suggesting an origin predating the radiation of eukaryotic lineages. Further, whilst we detect distant sequence relations between GARP, COG, exocyst and DSL1 components, these similarities most likely reflect convergent evolution of similar secondary structural elements. No similarity is found between the TRAPP and HOPS complexes and the other tethering factors. Overall, our data favour independent origins for the various tethering complexes. The taxa examined possess at least one homologue of each of the four SM protein families; since the four monophyletic families each encompass a wide diversity of eukaryotes, the SM protein families very likely evolved before the last common

  5. A dual-tagging system for the affinity purification of mammalian protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Giannone, Richard J [ORNL; McDonald, W Hayes [ORNL; Hurst, Gregory {Greg} B [ORNL; Huang, Ying [ORNL; Wu, Jun [ORNL; Liu, Yie [National Institute on Aging, Baltimore; Wang, Yisong [ORNL

    2007-01-01

    One popular method to elucidate protein-protein interactions involves the native co-purification of an affinity tagged protein and its interacting partners, which are subsequently identified through mass spectrometry (MS) (1). Although straightforward, reproducible, and broadly employed, this strategy is hampered by the efficacy of protein recoveries both in terms of sensitivity and specificity. This is especially pertinent to methodologies that employ a single-step of purification, where suboptimal enrichment of the bait protein and its partners over background can lead to masking of their signals. Although improvements to MS instrumentation generally increase peptide detection sensitivities, the problem of specificity, i.e. distinguishing specific from non-specific interacting proteins, remains. Thus ultimately, the limiting factor in the identification of specific interacting proteins lies with the purification itself. An effort to resolve this specificity issue has been made with the introduction of the Tandem Affinity Purification (TAP) tag. This construct consists of an IgG-binding domain and calmodulin binding peptide domain separated by a tobacco etch virus (TEV) protease cleavage site (2). The TAP method was originally developed in yeast and has best demonstrated its utility in the systematic identification of numerous multiprotein complexes in the yeast proteome (3). Although modifications to the original TAP have been successful in examining the protein networks of mammalian cells (4-7), the strategy offers a relatively low yield of bait and specific interacting proteins (8), and the success rate are usually on case-by-case basis. In addition, problems inherent to any protein tagging strategy remain, such as variable exposure of the affinity tag, disruption of the bait protein's ability to fold properly, steric exclusion of interacting partners, and/or ectopic overexpression of the fusion protein, which can lead to complications in both the

  6. Analysis of RNA-Protein Complexes by RNA Coimmunoprecipitation and RT-PCR Analysis from Caenorhabditis elegans

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Britta Jedamzik and Christian R. Eckmann1 Corresponding author (). ### INTRODUCTION RNA coimmunoprecipitation (co-IP) experiments are an extension of protein co-IP experiments in which in vivo RNA-protein complexes are investigated. This protocol describes how to perform RNA co-IPs from *C. elegans* whole-worm extracts. In principle, a protein-specific antibody is used to purify the protein of choice and its associated complex members from worm extract....

  7. Unique Role for the UbL-UbA Protein Ddi1 in Turnover of SCFUfo1 Complexes

    OpenAIRE

    Ivantsiv, Yelena; Kaplun, Ludmila; Tzirkin-Goldin, Regina; Shabek, Nitzan; Raveh, Dina

    2006-01-01

    SCF complexes are E3 ubiquitin-protein ligases that mediate degradation of regulatory and signaling proteins and control G1/S cell cycle progression by degradation of G1 cyclins and the cyclin-dependent kinase inhibitor, Sic1. Interchangeable F-box proteins bind the core SCF components; each recruits a specific subset of substrates for ubiquitylation. The F-box proteins themselves are rapidly turned over by autoubiquitylation, allowing rapid recycling of SCF complexes. Here we report a role f...

  8. A role for adaptor protein-3 complex in the organization of the endocytic pathway in Dictyostelium.

    Science.gov (United States)

    Charette, Steve J; Mercanti, Valentina; Letourneur, François; Bennett, Nelly; Cosson, Pierre

    2006-11-01

    Dictyostelium discoideum cells continuously internalize extracellular material, which accumulates in well-characterized endocytic vacuoles. In this study, we describe a new endocytic compartment identified by the presence of a specific marker, the p25 protein. This compartment presents features reminiscent of mammalian recycling endosomes: it is localized in the pericentrosomal region but distinct from the Golgi apparatus. It specifically contains surface proteins that are continuously endocytosed but rapidly recycled to the cell surface and thus absent from maturing endocytic compartments. We evaluated the importance of each clathrin-associated adaptor complex in establishing a compartmentalized endocytic system by studying the phenotype of the corresponding mutants. In knockout cells for mu3, a subunit of the AP-3 clathrin-associated complex, membrane proteins normally restricted to p25-positive endosomes were mislocalized to late endocytic compartments. Our results suggest that AP-3 plays an essential role in the compartmentalization of the endocytic pathway in Dictyostelium. PMID:17010123

  9. Protein biomarker enrichment by biomarker antibody complex elution for immunoassay biosensing.

    Science.gov (United States)

    Sabatte, Gwenola; Feitsma, Harma; Evers, Toon H; Prins, Menno W J

    2011-11-15

    It is very challenging to perform sample enrichment for protein biomarkers because proteins can easily change conformation and denature. In this paper we demonstrate protein enrichment suited for high-sensitivity integrated immuno-biosensing. The method enhances the concentration of the biomarkers and simultaneously removes matrix components that could interfere with the immunoassay. Biomarkers are captured using antibody coated magnetic particles and the biomarker antibody complexes are released by enzymatic elution. The eluted complexes are subsequently detected in a sandwich immunoassay biosensor. A scaling study of the enrichment process demonstrates an enrichment factor of 15 in buffer and plasma. We analyze the enrichment factor in terms of the three basic steps of the assay (capture, concentration, elution) and we quantify their respective efficiencies. The process is suited for integration into bio-analytical tools.

  10. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael;

    2007-01-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly...... propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins....... identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to...

  11. Nanopatterned Protein Films Directed by Ionic Complexation with Water-Soluble Diblock Copolymers.

    Science.gov (United States)

    Kim, Bokyung; Lam, Christopher N; Olsen, Bradley D

    2012-06-12

    The use of ionic interactions to direct both protein templating and block copolymer self-assembly into nanopatterned films with only aqueous processing conditions is demonstrated using block copolymers containing both thermally responsive and pH responsive blocks. Controlled reversible addition-fragmentation chain-transfer (RAFT) polymerization is employed to synthesize poly(N-isopropylacrylamide-b-2-(dimethylamino)ethyl acrylate) (PNIPAM-b-PDMAEA) diblock copolymers. The pH-dependent ionic complexation between the fluorescent protein, mCherry, and the ionic PDMAEA block is established using dynamic light scattering (DLS) and UV-Vis spectroscopy. DLS shows that the size of the resulting coacervate micelles depends strongly on pH, while UV-Vis spectroscopy shows a correlation between the protein's absorption maximum and the ionic microenvironment. Zeta potential measurements clearly indicate the ionic nature of the complex-forming interactions. Spin casting was used to prepare nanostructured films from the protein-block copolymer coacervates. After film formation, the lower critical solution temperature (LCST) of the PNIPAM blocks allows the nanomaterial to be effectively immobilized in aqueous environments at physiological temperatures, enabling potential use as a controlled protein release material or polymer matrix for protein immobilization. At pH 9.2 and 7.8, the release rates are at least 10 times faster than that at pH 6.4 due to weaker interaction between protein and PNIPAM-b-PDMAEA (PND) diblock copolymer. Due to the ionic environment in which protein is confined, the majority of the protein (80%) remains active, independent of pH, even after having been dehydrated in vacuum and confined in the films. PMID:24904186

  12. TIMAP-protein phosphatase 1-complex controls endothelin-1 production via ECE-1 dephosphorylation.

    Science.gov (United States)

    Boratkó, Anita; Veréb, Zoltán; Petrovski, Goran; Csortos, Csilla

    2016-04-01

    Endothelin induced signaling pathways can affect blood pressure and vascular tone, but the influence of endothelins on tumor cells is also significant. We have detected elevated endothelin-1 secretion from TIMAP (TGF-β inhibited membrane associated protein) depleted vascular endothelial cells. The autocrine signaling activated by the elevated endothelin-1 level through the ETB receptors evoked an angiogenic-like phenotype, the cells assumed an elongated morphology, and enhanced tube formation and wound healing abilities. The depleted protein, TIMAP, is a highly specific and abundant protein in the endothelial cells, and it is a regulatory/targeting subunit for the catalytic subunit of protein phosphatase 1 (PP1c). Protein-protein interaction between the TIMAP-PP1c complex and the endothelin converting enzyme-1 (ECE-1) was detected, the latter of which is a transmembrane protein that produces the biologically active 21-amino acid form of endothelin-1 from proendothelin. The results indicate that silencing of TIMAP induces a reduction in TIMAP-PP1c activity connected to ECE-1. This leads to an increase in the amount of ECE-1 protein in the plasma membrane and a consequent increase in endothelin-1 secretion. Similarly, activation of PKC, the kinase responsible for ECE-1 phosphorylation increased ECE-1 protein level in the membrane fraction of the endothelial cells. The elevated ECE-1 level was mitigated in time in normal cells, but was clearly preserved in TIMAP-depleted cells. Overall, our results indicate that PKC-phosphorylated ECE-1 is a TIMAP-PP1c substrate and this phosphatase complex has an important role in endothelin-1 production of EC through the regulation of ECE-1 activity.

  13. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death. PMID:26561036

  14. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  15. VS-APPLE: A Virtual Screening Algorithm Using Promiscuous Protein-Ligand Complexes.

    Science.gov (United States)

    Okuno, Tatsuya; Kato, Koya; Terada, Tomoki P; Sasai, Masaki; Chikenji, George

    2015-06-22

    As the number of structurally resolved protein-ligand complexes increases, the ligand-binding pockets of many proteins have been found to accommodate multiple different compounds. Effective use of these structural data is important for developing virtual screening (VS) methods that identify bioactive compounds. Here, we introduce a VS method, VS-APPLE (Virtual Screening Algorithm using Promiscuous Protein-Ligand complExes), based on promiscuous protein-ligand binding structures. In VS-APPLE, multiple ligands bound to a pocket are combined into a query template for screening. Both the structural match between a test compound and the multiple-ligand template and the possible collisions between the test compound and the target protein are evaluated by an efficient geometric hashing method. The performance of VS-APPLE was examined on a filtered, clustered version of the Directory of Useful Decoys data set. In Area Under the Curve analyses of this data set, VS-APPLE outperformed several popular screening programs. Judging from the performance of VS-APPLE, the structural data of promiscuous protein-ligand bindings could be further analyzed and exploited for developing VS methods.

  16. X-ray structure of the mammalian GIRK2-βγ G-protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Whorton, Matthew R.; MacKinnon, Roderick [Rockefeller

    2013-07-30

    G-protein-gated inward rectifier K+ (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K+ channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G-protein subunits at the interfaces between four K+ channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with ‘membrane delimited’ activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) and intracellular Na+ ions participate in multi-ligand regulation of GIRK channels.

  17. Validation of a new restraint docking method for solution structure determinations of protein-ligand complexes

    Energy Technology Data Exchange (ETDEWEB)

    Polshakov, Vladimir I.; Morgan, William D.; Birdsall, Berry; Feeney, James [National Institute for Medical Research, Division of Molecular Structure (United Kingdom)

    1999-06-15

    A new method is proposed for docking ligands into proteins in cases where an NMR-determined solution structure of a related complex is available. The method uses a set of experimentally determined values for protein-ligand, ligand-ligand, and protein-protein restraints for residues in or near to the binding site, combined with a set of protein-protein restraints involving all the other residues which is taken from the list of restraints previously used to generate the reference structure of a related complex. This approach differs from ordinary docking methods where the calculation uses fixed atomic coordinates from the reference structure rather than the restraints used to determine the reference structure. The binding site residues influenced by replacing the reference ligand by the new ligand were determined by monitoring differences in {sup 1}H chemical shifts. The method has been validated by showing the excellent agreement between structures of L. casei dihydrofolate reductase.trimetrexate calculated by conventional methods using a full experimentally determined set of restraints and those using this new restraint docking method based on an L. casei dihydrofolate reductase.methotrexate reference structure.

  18. Proteins with complex architecture as potential targets for drug design: a case study of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Bálint Mészáros

    2011-07-01

    Full Text Available Lengthy co-evolution of Homo sapiens and Mycobacterium tuberculosis, the main causative agent of tuberculosis, resulted in a dramatically successful pathogen species that presents considerable challenge for modern medicine. The continuous and ever increasing appearance of multi-drug resistant mycobacteria necessitates the identification of novel drug targets and drugs with new mechanisms of action. However, further insights are needed to establish automated protocols for target selection based on the available complete genome sequences. In the present study, we perform complete proteome level comparisons between M. tuberculosis, mycobacteria, other prokaryotes and available eukaryotes based on protein domains, local sequence similarities and protein disorder. We show that the enrichment of certain domains in the genome can indicate an important function specific to M. tuberculosis. We identified two families, termed pkn and PE/PPE that stand out in this respect. The common property of these two protein families is a complex domain organization that combines species-specific regions, commonly occurring domains and disordered segments. Besides highlighting promising novel drug target candidates in M. tuberculosis, the presented analysis can also be viewed as a general protocol to identify proteins involved in species-specific functions in a given organism. We conclude that target selection protocols should be extended to include proteins with complex domain architectures instead of focusing on sequentially unique and essential proteins only.

  19. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

    Science.gov (United States)

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-08-12

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

  20. Drosophila Torsin Protein Regulates Motor Control and Stress Sensitivity and Forms a Complex with Fragile-X Mental Retardation Protein

    Science.gov (United States)

    Ahn, Hyo-Min; Koh, Young Ho

    2016-01-01

    We investigated unknown in vivo functions of Torsin by using Drosophila as a model. Downregulation of Drosophila Torsin (DTor) by DTor-specific inhibitory double-stranded RNA (RNAi) induced abnormal locomotor behavior and increased susceptibility to H2O2. In addition, altered expression of DTor significantly increased the numbers of synaptic boutons. One important biochemical consequence of DTor-RNAi expression in fly brains was upregulation of alcohol dehydrogenase (ADH). Altered expression of ADH has also been reported in Drosophila Fragile-X mental retardation protein (DFMRP) mutant flies. Interestingly, expression of DFMRP was altered in DTor mutant flies, and DTor and DFMRP were present in the same protein complexes. In addition, DTor and DFMRP immunoreactivities were partially colocalized in several cellular organelles in larval muscles. Furthermore, there were no significant differences between synaptic morphologies of dfmrp null mutants and dfmrp mutants expressing DTor-RNAi. Taken together, our evidences suggested that DTor and DFMRP might be present in the same signaling pathway regulating synaptic plasticity. In addition, we also found that human Torsin1A and human FMRP were present in the same protein complexes, suggesting that this phenomenon is evolutionarily conserved. PMID:27313903

  1. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

    Science.gov (United States)

    Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Vercruysse, Leen; Dedecker, Maarten; Verkest, Aurine; Vandepoele, Klaas; Martens, Lennart; Witters, Erwin; Gevaert, Kris; De Jaeger, Geert

    2015-01-01

    Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

  2. Phenanthrene binding by humic acid–protein complexes as studied by passive dosing technique

    International Nuclear Information System (INIS)

    This work investigated the binding behavior of phenanthrene by humic acids (HA-2 and HA-5), proteins (bovine serum albumin (BSA)), lysozyme and pepsin), and their complexes using a passive dosing technique. All sorption isotherms were fitted well with Freundlich model and the binding capability followed an order of HA-5 > HA-2 > BSA > pepsin > lysozyme. In NaCl solution, phenanthrene binding to HA-BSA complexes was much higher than the sum of binding to individual HA and BSA, while there was no enhancement for HA-pepsin. Positively charged lysozyme slightly lowered phenanthrene binding on both HAs due to strong aggregation of HA-lysozyme complexes, leading to reduction in the number of binding sites. The binding enhancement by HA-BSA was observed under all tested ion species and ionic strengths. This enhancement can be explained by unfolding of protein, reduction of aggregate size and formation of HA-BSA complexes with favorable conformations for binding phenanthrene. Highlights: • Phenanthrene binding capability followed an order: HA-5>HA-2>BSA>pepsin>lysozyme. • Phenanthrene binding to HA-BSA was enhanced relative to individual HA and BSA. • Binding enhancement to HA-BSA was observed under all tested solution conditions. • The enhancement is related to BSA unfolding, size reduction and HA-BSA complexation. -- Phenanthrene binding to HA-BSA complexes is much higher than the sum to individual HA and BSA while there was no binding enhancement to HA-pepsin or HA-lysozyme

  3. Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein: A common step in genetic recombination

    OpenAIRE

    Kantake, Noriko; Madiraju, Murty V. V. M.; Sugiyama, Tomohiko; Kowalczykowski, Stephen C.

    2002-01-01

    We present biochemical evidence for the functional similarity of Escherichia coli RecO protein and bacteriophage T4 UvsY protein to eukaryotic Rad52 protein. Although Rad52 protein is conserved in eukaryotes, no sequence homologue has been found in prokaryotes or archeabacteria. Rad52 protein has two unique activities: facilitation of replication protein-A (RPA) displacement by Rad51 protein and annealing of RPA–single-stranded DNA (ssDNA) complexes. Both activities require species-specific i...

  4. Prediction of protein structural features from sequence data based on Shannon entropy and Kolmogorov complexity.

    Science.gov (United States)

    Bywater, Robert Paul

    2015-01-01

    While the genome for a given organism stores the information necessary for the organism to function and flourish it is the proteins that are encoded by the genome that perhaps more than anything else characterize the phenotype for that organism. It is therefore not surprising that one of the many approaches to understanding and predicting protein folding and properties has come from genomics and more specifically from multiple sequence alignments. In this work I explore ways in which data derived from sequence alignment data can be used to investigate in a predictive way three different aspects of protein structure: secondary structures, inter-residue contacts and the dynamics of switching between different states of the protein. In particular the use of Kolmogorov complexity has identified a novel pathway towards achieving these goals.

  5. Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons.

    Science.gov (United States)

    Benleulmi-Chaachoua, Abla; Chen, Lina; Sokolina, Kate; Wong, Victoria; Jurisica, Igor; Emerit, Michel Boris; Darmon, Michèle; Espin, Almudena; Stagljar, Igor; Tafelmeyer, Petra; Zamponi, Gerald W; Delagrange, Philippe; Maurice, Pascal; Jockers, Ralf

    2016-01-01

    In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT1 and MT2 receptors, belonging to the G protein-coupled receptor (GPCR) super-family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT1 and MT2 receptors, which comprises 378 individual proteins. Among the proteins interacting with MT1 , but not with MT2 , we identified several presynaptic proteins, suggesting a potential role of MT1 in neurotransmission. Presynaptic localization of MT1 receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT1 physically interacts with the voltage-gated calcium channel Cav 2.2 and inhibits Cav 2.2-promoted Ca(2+) entry in an agonist-independent manner. In conclusion, we show that MT1 is part of the presynaptic protein network and negatively regulates Cav 2.2 activity, providing a first hint for potential synaptic functions of MT1. PMID:26514267

  6. Toll-Like Receptor Signaling in Vertebrates: Testing the Integration of Protein, Complex, and Pathway Data in the Protein Ontology Framework

    OpenAIRE

    Cecilia Arighi; Veronica Shamovsky; Anna Maria Masci; Alan Ruttenberg; Barry Smith; Natale, Darren A.; Cathy Wu; Peter D'Eustachio

    2015-01-01

    The Protein Ontology (PRO) provides terms for and supports annotation of species-specific protein complexes in an ontology framework that relates them both to their components and to species-independent families of complexes. Comprehensive curation of experimentally known forms and annotations thereof is expected to expose discrepancies, differences, and gaps in our knowledge. We have annotated the early events of innate immune signaling mediated by Toll-Like Receptor 3 and 4 complexes in hum...

  7. Molecular simulation-based structural prediction of protein complexes in mass spectrometry: the human insulin dimer.

    Science.gov (United States)

    Li, Jinyu; Rossetti, Giulia; Dreyer, Jens; Raugei, Simone; Ippoliti, Emiliano; Lüscher, Bernhard; Carloni, Paolo

    2014-09-01

    Protein electrospray ionization (ESI) mass spectrometry (MS)-based techniques are widely used to provide insight into structural proteomics under the assumption that non-covalent protein complexes being transferred into the gas phase preserve basically the same intermolecular interactions as in solution. Here we investigate the applicability of this assumption by extending our previous structural prediction protocol for single proteins in ESI-MS to protein complexes. We apply our protocol to the human insulin dimer (hIns2) as a test case. Our calculations reproduce the main charge and the collision cross section (CCS) measured in ESI-MS experiments. Molecular dynamics simulations for 0.075 ms show that the complex maximizes intermolecular non-bonded interactions relative to the structure in water, without affecting the cross section. The overall gas-phase structure of hIns2 does exhibit differences with the one in aqueous solution, not inferable from a comparison with calculated CCS. Hence, care should be exerted when interpreting ESI-MS proteomics data based solely on NMR and/or X-ray structural information. PMID:25210764

  8. Molecular simulation-based structural prediction of protein complexes in mass spectrometry: the human insulin dimer.

    Directory of Open Access Journals (Sweden)

    Jinyu Li

    2014-09-01

    Full Text Available Protein electrospray ionization (ESI mass spectrometry (MS-based techniques are widely used to provide insight into structural proteomics under the assumption that non-covalent protein complexes being transferred into the gas phase preserve basically the same intermolecular interactions as in solution. Here we investigate the applicability of this assumption by extending our previous structural prediction protocol for single proteins in ESI-MS to protein complexes. We apply our protocol to the human insulin dimer (hIns2 as a test case. Our calculations reproduce the main charge and the collision cross section (CCS measured in ESI-MS experiments. Molecular dynamics simulations for 0.075 ms show that the complex maximizes intermolecular non-bonded interactions relative to the structure in water, without affecting the cross section. The overall gas-phase structure of hIns2 does exhibit differences with the one in aqueous solution, not inferable from a comparison with calculated CCS. Hence, care should be exerted when interpreting ESI-MS proteomics data based solely on NMR and/or X-ray structural information.

  9. Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes.

    Directory of Open Access Journals (Sweden)

    Justin D Blethrow

    Full Text Available The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI and electrospray ionization (ESI ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.

  10. Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

    Energy Technology Data Exchange (ETDEWEB)

    Pröpper, Kevin [University of Göttingen, (Germany); Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Meindl, Kathrin; Sammito, Massimo [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Dittrich, Birger; Sheldrick, George M. [University of Göttingen, (Germany); Pohl, Ehmke, E-mail: ehmke.pohl@durham.ac.uk [Durham University, (United Kingdom); Usón, Isabel, E-mail: ehmke.pohl@durham.ac.uk [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Institucio Catalana de Recerca i Estudis Avancats (ICREA), (Spain); University of Göttingen, (Germany)

    2014-06-01

    The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

  11. Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate.

    LENUS (Irish Health Repository)

    O'Connor, Roisin

    2010-01-01

    Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.

  12. FORMATION AND QUANTIFICATION OF PROTEIN COMPLEXES BETWEEN PEROXISOMAL ALCOHOL OXIDASE AND GROEL

    OpenAIRE

    EVERS, ME; Langer, T.; Harder, W; HARTL, FU; Veenhuis, M; Hartl, Franz-Ulrich

    1992-01-01

    We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition of GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels. In the presence of MgAMP-PNP or MgADP the affinity of Gro...

  13. Proximity probing assays for simultaneous visualization of protein complexes in situ

    DEFF Research Database (Denmark)

    Moreira, José; Thorsen, Stine Buch; Brünner, Nils;

    2013-01-01

    EVALUATION OF: Leuchowius KJ, Clausson CM, Grannas K et al. Parallel visualization of multiple protein complexes in individual cells in tumor tissue. Mol. Cell Proteomics doi:10.1074/mcp.O112.023374 (2013) (Epub ahead of print). Techniques for in situ detection and quantification of proteins...... in fixed tissue remain an important element of both basic biological analyses and clinical biomarker research. The practical importance of such techniques can be exemplified by the everyday clinical use of immunohistochemical detection of the estrogen receptor and HER2 in tissues from breast cancer...

  14. A COMPLEX OF INTERMEDIATE FILAMENT PROTEIN-DNA: A TARGET FOR AUTOANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS?

    Institute of Scientific and Technical Information of China (English)

    阎锡德; S.Kuhn; P.Traub

    1995-01-01

    Autoantibodies in systemic lupus erythematosus which cross-react with double stranded DNA and in-termediate filament proteins are frequently reported.However,little is Known about the origin and the target of these antibodies.In this paper,a polyspecific monoclonal antibody,XY12,produced by the im-munization of genetically non-autoimmune mice with a DNA-protein complex is detsiled.Its antigen bind-ing patterms are very similar to the autoantibodies.The data suggest that these autoantibodies may be trig-gered by a circulating nucleoprotein.

  15. Survey of large protein complexes D. vulgaris reveals great structural diversity

    Energy Technology Data Exchange (ETDEWEB)

    Han, B.-G.; Dong, M.; Liu, H.; Camp, L.; Geller, J.; Singer, M.; Hazen, T. C.; Choi, M.; Witkowska, H. E.; Ball, D. A.; Typke, D.; Downing, K. H.; Shatsky, M.; Brenner, S. E.; Chandonia, J.-M.; Biggin, M. D.; Glaeser, R. M.

    2009-08-15

    An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr {approx} 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate {approx}10 times greater than that of previous 'proteomic' screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

  16. Effects of conformational ordering on protein/polyelectrolyte electrostatic complexation: ionic binding and chain stiffening

    Science.gov (United States)

    Cao, Yiping; Fang, Yapeng; Nishinari, Katsuyoshi; Phillips, Glyn O.

    2016-01-01

    Coupling of electrostatic complexation with conformational transition is rather general in protein/polyelectrolyte interaction and has important implications in many biological processes and practical applications. This work studied the electrostatic complexation between κ-carrageenan (κ-car) and type B gelatin, and analyzed the effects of the conformational ordering of κ-car induced upon cooling in the presence of potassium chloride (KCl) or tetramethylammonium iodide (Me4NI). Experimental results showed that the effects of conformational ordering on protein/polyelectrolyte electrostatic complexation can be decomposed into ionic binding and chain stiffening. At the initial stage of conformational ordering, electrostatic complexation can be either suppressed or enhanced due to the ionic bindings of K+ and I− ions, which significantly alter the charge density of κ-car or occupy the binding sites of gelatin. Beyond a certain stage of conformational ordering, i.e., helix content θ > 0.30, the effect of chain stiffening, accompanied with a rapid increase in helix length ζ, becomes dominant and tends to dissociate the electrostatic complexation. The effect of chain stiffening can be theoretically interpreted in terms of double helix association. PMID:27030165

  17. Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3'-Terminal Fragment of 16S rRNA in E. coli.

    Science.gov (United States)

    Golovin, A V; Khayrullina, G A; Kraal, B; Kopylov, Capital A Cyrillic М

    2012-10-01

    For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA-protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA-protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV-induced RNA-protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA- protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA - protein cross-link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA-protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

  18. Determination of the stoichiometry, structure, and distribution in living cells of protein complexes from analysis of single-molecular-complexes FRET

    Science.gov (United States)

    Stoneman, Michael R.; Patowary, S.; Roesch, M. T.; Singh, D. R.; Strogolov, V.; Oliver, J. A.; Raicu, V.

    2011-03-01

    Advances in two-photon microscopy with spectral resolution (TPM-SR) and the development of a simple theory of Förster Resonance Energy Transfer (FRET) for single molecular complexes recently lead to the development of a novel method for the determination of structure and localization in living cells of membrane protein complexes (Raicu et al., Nature Photon., 3, 2009). An appealing feature of this method is its ability to provide such important information while being unaffected by spurious signals originating from stochastic FRET (Singh and Raicu, Biophys. J., 98, 2010). We will present the results obtained from our recent studies of trimeric FRET calibration standards expressed in the cytoplasm of Chinese hamster ovary (CHO) cells, as well as a model G protein-coupled receptor expressed in the membrane of yeast. Emphasis will be placed on the measurement and analysis of single-molecular-complex FRET data for determination of the quaternary structure of some proteins (or the protein complex structure).

  19. Abstract 5324: Pan-cancer identification of mutated pathways and protein complexes

    DEFF Research Database (Denmark)

    Leiserson, Mark D.; Vandin, Fabio; Wu, Hsin-Ta;

    2014-01-01

    -exome sequencing and copy number aberration data from 3299 samples of twelve tumor types from TCGA Pan-Cancer project. Both algorithms identified gene sets that overlap well-known cancer pathways (e.g. TP53, MAPK, and RAS signaling pathways), as well as genes and complexes with less characterized roles in cancer...... of tumors, prioritizing genes and mutations in the long tail for further experimental studies.Citation Format: Mark D. Leiserson, Fabio Vandin, Hsin-Ta Wu, Jason R. Dobson, Benjamin R. Raphael. Pan-cancer identification of mutated pathways and protein complexes. [abstract]. In: Proceedings of the 105th...

  20. Physicochemical Characterization and Potential Prebiotic Effect of Whey Protein Isolate/Inulin Nano Complex.

    Science.gov (United States)

    Ha, Ho-Kyung; Jeon, Na-Eun; Kim, Jin Wook; Han, Kyoung-Sik; Yun, Sung Seob; Lee, Mee-Ryung; Lee, Won-Jae

    2016-01-01

    The purposes of this study were to investigate the impacts of concentration levels of whey protein isolate (WPI) and inulin on the formation and physicochemical properties of WPI/inulin nano complexes and to evaluate their potential prebiotic effects. WPI/inulin nano complexes were produced using the internal gelation method. Transmission electron microscopy (TEM) and particle size analyzer were used to assess the morphological and physicochemical characterizations of nano complexes, respectively. The encapsulation efficiency of resveratrol in nano complexes was studied using HPLC while the potential prebiotic effects were investigated by measuring the viability of probiotics. In TEM micrographs, the globular forms of nano complexes in the range of 10 and 100 nm were successfully manufactured. An increase in WPI concentration level from 1 to 3% (w/v) resulted in a significant (pcomplexs while inulin concentration level did not affect the size of nano complexes. The polydispersity index of nano complexes was below 0.3 in all cases while the zeta-potential values in the range of -2 and -12 mV were observed. The encapsulation efficiency of resveratrol was significantly (pcomplexes exhibited similar viability of probiotics with free inulin and had significantly (pcomplexes and had potential prebiotic effect. PMID:27194937

  1. Casein kinase Ⅱ interacts with prion protein in vitro and forms complex with na-tive prion protein in vivo

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein to pathologic isoform. Casein kinase Ⅱ (CK2) is a ubiquitously expressed and evolutiouarily conserved pleiotropic protein kinase that is essential for viability. To explore the possible molecular interaction between CK2 and prion protein (PrP), the full-length sequences of human CK2α and CK2β complementary DNA were amplified with reverse transcription-polymerase chain reaction using the total messenger RNA from cell line SH-SY5Y as the template; then, the fusion proteins histidine-CK2α and glutathione S-transferase-histidine-CK2β were expressed in Escherichia coll. The interaction between CK2 and PrP was evaluated with co-immunoprecipi-tation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2α, but not with CK2β. The native CK2 and PrP in hamster brains interacted with each other, forming protein complexes. Three different glycosylated forms of PrP (diglycosylated, monoglycosylated and unglycosylated PrP) from normal brains interacted with the CK2α subunit, though the unglycosylated PrP seemed to have a stronger binding ability with CK2α subunit. The domain responsible for interacting with CK2α was located at the C-terminal segment of PrP (residues 91-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2α (both in recombinant and native categories), scientific clues for further assessing the potential biological significance of the PrP-CK2 interaction, and the possible role of CK2 in the pathogenesis of prion diseases.

  2. Versatile annotation and publication quality visualization of protein complexes using POLYVIEW-3D

    Directory of Open Access Journals (Sweden)

    Meller Jaroslaw

    2007-08-01

    Full Text Available Abstract Background Macromolecular visualization as well as automated structural and functional annotation tools play an increasingly important role in the post-genomic era, contributing significantly towards the understanding of molecular systems and processes. For example, three dimensional (3D models help in exploring protein active sites and functional hot spots that can be targeted in drug design. Automated annotation and visualization pipelines can also reveal other functionally important attributes of macromolecules. These goals are dependent on the availability of advanced tools that integrate better the existing databases, annotation servers and other resources with state-of-the-art rendering programs. Results We present a new tool for protein structure analysis, with the focus on annotation and visualization of protein complexes, which is an extension of our previously developed POLYVIEW web server. By integrating the web technology with state-of-the-art software for macromolecular visualization, such as the PyMol program, POLYVIEW-3D enables combining versatile structural and functional annotations with a simple web-based interface for creating publication quality structure rendering, as well as animated images for Powerpoint™, web sites and other electronic resources. The service is platform independent and no plug-ins are required. Several examples of how POLYVIEW-3D can be used for structural and functional analysis in the context of protein-protein interactions are presented to illustrate the available annotation options. Conclusion POLYVIEW-3D server features the PyMol image rendering that provides detailed and high quality presentation of macromolecular structures, with an easy to use web-based interface. POLYVIEW-3D also provides a wide array of options for automated structural and functional analysis of proteins and their complexes. Thus, the POLYVIEW-3D server may become an important resource for researches and educators in

  3. Report on the research conducted under the funding of the Sloan foundation postdoctoral fellowship in Computational Molecular Biology [Systematic study of protein-protein complexes] Final report

    Energy Technology Data Exchange (ETDEWEB)

    Sheinerman, Felix

    2001-06-01

    A central question in molecular biology is what structural features are common at protein-protein interfaces and what energetic factors define the affinity and specificity of protein-protein association. Analysis of structural and mutational data on protein-protein interfaces revealed that protein-protein interfaces of different functional classes contain many more energetically important charged and polar residues than was previously thought. Since, in the context of protein folding studies, polar interactions are believed to destabilize the folded proteins, this observation raised the question as to the forces that determine the stability of protein complexes. To investigate this issue in detail, the authors developed a number of partitioning schemes that allowed them to investigate the role of selected residues, ion pairs, and networks of polar interactions in protein-protein association. The methods developed were applied to the analysis of four different protein-protein interfaces: the ribonuclease barnase and its inhibitor barstar, the human growth hormone and its receptor, subtype N9 influenze virus neuraminidase and NC41 antibody, and the Ras Binding Domain of kinase cRaf and a Ras homologue Rap1A. The calculations revealed a surprising variability in how polar interactions affect the stability of different complexes. The finding that positions of charged and polar residues on protein-protein interfaces are optimized with respect to electrostatic interactions suggests that this property can be employed for the discrimination between native conformations and trial complexes generated by a docking algorithm. Analysis indicated the presence of SH2 domains in Janus family of non-receptor protein tyrosine kinases.

  4. Radiation quality effects on pigment-protein complex of maize and pine

    International Nuclear Information System (INIS)

    Maize hybrid ZP-704 and Pinus nigra seedlings were grown under the same low irradiance (2.0 µmol/s m²) of white (WR), blue (BR) or red (RR) irradiation and adapted similarly. Radiation quality differences during chloroplast development had a greater effect on the ratio of PSII:PSI complexes than on chlorophyll (Chl) a:b ratio. RR in mesophyll chloroplasts induced primarily an increased accumulation of Chl a, b, xanthophylls, light-harvesting complex proteins LHC1 and LHC3, and PSII-bound polypeptides. BR-treated plants were more efficient in the synthesis of β-carotene, Chl-proteins and PSI-bound polypeptides. BR resulted in the production of sun type chloroplasts while RR gave shade type chloroplasts and WR resulted in intermediate chloroplasts

  5. Role of pigment-protein coupling and pathways of excitation energy transfer in FMO complex

    CERN Document Server

    Singh, Davinder

    2016-01-01

    We theoretically investigate the effect of different pigment-protein couplings and the role of quantum interference among different energy transfer channels in excitation energy transfer (EET) in FMO complex. We employ the non-Markovian master equation that allows the use of different values of pigment-protein couplings and cut-off frequencies for different BChla sites, in the adiabatic limit of electron transfer in FMO complex. Several pathways of EET are identified and investigated using a realistic set of pigment-pigment couplings and the site energy of each BChla site. We analyze that it is the destructive interference between different channels of a particular pathway that is responsible for the time-scales of oscillations of excitation energy as observed in the recent experiments.

  6. Different types of nsP3-containing protein complexes in Sindbis virus-infected cells.

    Science.gov (United States)

    Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

    2008-10-01

    Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

  7. Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells▿

    Science.gov (United States)

    Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

    2008-01-01

    Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions. PMID:18684830

  8. The PAM domain, a multi-protein complex-associated module with an all-alpha-helix fold

    Directory of Open Access Journals (Sweden)

    Izaurralde Elisa

    2003-12-01

    Full Text Available Abstract Background Multimeric protein complexes have a role in many cellular pathways and are highly interconnected with various other proteins. The characterization of their domain composition and organization provides useful information on the specific role of each region of their sequence. Results We identified a new module, the PAM domain (PCI/PINT associated module, present in single subunits of well characterized multiprotein complexes, like the regulatory lid of the 26S proteasome, the COP-9 signalosome and the Sac3-Thp1 complex. This module is an around 200 residue long domain with a predicted TPR-like all-alpha-helical fold. Conclusions The occurrence of the PAM domain in specific subunits of multimeric protein complexes, together with the role of other all-alpha-helical folds in protein-protein interactions, suggest a function for this domain in mediating transient binding to diverse target proteins.

  9. Transition fibre protein FBF1 is required for the ciliary entry of assembled intraflagellar transport complexes

    OpenAIRE

    WEI, QING; Xu, Qingwen; Zhang, Yuxia; Li, Yujie; Zhang, Qing; Hu, Zeng; Harris, Peter C.; Torres, Vicente E.; Ling, Kun; Hu, Jinghua

    2013-01-01

    Sensory organelle cilia play critical roles in mammalian embryonic development and tissue homeostasis. Intraflagellar transport (IFT) machinery is required for the assembly and maintenance of cilia. Yet how this large complex passes through the size-dependent barrier at the ciliary base remains enigmatic. Here we report that FBF1, a highly conserved transition fibre protein, is required for the ciliary import of assembled IFT particles at the cilia base. We cloned dyf-19, the C. elegans homol...

  10. Trapping electrophoresis and ratchets: a theoretical study for DNA-protein complexes.

    OpenAIRE

    Desruisseaux, C; Slater, G W; Kist, T B

    1998-01-01

    Recently, Griess and Serwer (1998. Biophys. J. 74:A71) showed that it was possible to use trapping electrophoresis and unbiased but asymmetrical electric field pulses to build a correlation ratchet that would allow the efficient separation of naked DNAs from identical DNAs that form a complex with a bulky object such as a protein. Here we present a theoretical investigation of this novel macromolecular separation process. We start by looking at the general features of this electrophoretic rat...

  11. Protoss: a holistic approach to predict tautomers and protonation states in protein-ligand complexes

    OpenAIRE

    Bietz, Stefan; Urbaczek, Sascha; Schulz, Benjamin; Rarey, Matthias

    2014-01-01

    The calculation of hydrogen positions is a common preprocessing step when working with crystal structures of protein-ligand complexes. An explicit description of hydrogen atoms is generally needed in order to analyze the binding mode of particular ligands or to calculate the associated binding energies. Due to the large number of degrees of freedom resulting from different chemical moieties and the high degree of mutual dependence this problem is anything but trivial. In addition to an effici...

  12. A Split-Ubiquitin Based Strategy Selecting for Protein Complex-Interfering Mutations.

    Science.gov (United States)

    Gronemeyer, Thomas; Chollet, Julian; Werner, Stefan; Glomb, Oliver; Bäuerle, Anne; Johnsson, Nils

    2016-01-01

    Understanding the topologies and functions of protein interaction networks requires the selective removal of single interactions. We introduce a selection strategy that enriches among a random library of alleles for mutations that impair the binding to a given partner protein. The selection makes use of a split-ubiquitin based protein interaction assay. This assay provides yeast cells that carry protein complex disturbing mutations with the advantage of being able to survive on uracil-lacking media. Applied to the exemplary interaction between the PB domains of the yeast proteins Bem1 and Cdc24, we performed two independent selections. The selections were either analyzed by Sanger sequencing of isolated clones or by next generation sequencing (NGS) of pools of clones. Both screens enriched for the same mutation in position 833 of Cdc24. Biochemical analysis confirmed that this mutation disturbs the interaction with Bem1 but not the fold of the protein. The larger dataset obtained by NGS achieved a more complete representation of the bipartite interaction interface of Cdc24.

  13. The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ruiying; Zheng, Han; Preamplume, Gan; Shao, Yaming; Li, Hong [FSU

    2012-03-15

    The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.

  14. An experimental strategy for the identification of AMPylation targets from complex protein samples.

    Science.gov (United States)

    Pieles, Kathrin; Glatter, Timo; Harms, Alexander; Schmidt, Alexander; Dehio, Christoph

    2014-05-01

    AMPylation is a posttranslational modification (PTM) that has recently caught much attention in the context of bacterial infections as pathogens were shown to secrete Fic proteins that AMPylate Rho GTPases and thus interfere with host cell signaling processes. Although Fic proteins are widespread and found in all kingdoms of life, only a small number of AMPylation targets are known to date. A major obstacle to target identification is the limited availability of generic strategies allowing sensitive and robust identification of AMPylation events. Here, we present an unbiased MS-based approach utilizing stable isotope-labeled ATP. The ATP isotopes are transferred onto target proteins in crude cell lysates by in vitro AMPylation introducing specific reporter ion clusters that allow detection of AMPylated peptides in complex biological samples by MS analysis. Applying this strategy on the secreted Fic protein Bep2 of Bartonella rochalimae, we identified the filamenting protein vimentin as an AMPylation target that was confirmed by independent assays. Vimentin represents a new class of target proteins and its identification emphasizes our method as a valuable tool to systematically uncover AMPylation targets. Furthermore, the approach can be generically adapted to study targets of other PTMs that allow incorporation of isotopically labeled substrates.

  15. Modification of the protein corona-nanoparticle complex by physiological factors.

    Science.gov (United States)

    Braun, Nicholas J; DeBrosse, Madeleine C; Hussain, Saber M; Comfort, Kristen K

    2016-07-01

    Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications.

  16. Proteomic Identification of Altered Cerebral Proteins in the Complex Regional Pain Syndrome Animal Model

    Directory of Open Access Journals (Sweden)

    Francis Sahngun Nahm

    2014-01-01

    Full Text Available Background. Complex regional pain syndrome (CRPS is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP model, a novel experimental model of CRPS. Materials and Methods. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Conclusion. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS.

  17. Utilization of Methyl Proton Resonances in Cross-Saturation Measurement for Determining the Interfaces of Large Protein-Protein Complexes

    International Nuclear Information System (INIS)

    Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220-223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of 1H2Ogreatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach

  18. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    Science.gov (United States)

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion.

  19. Subcritical Water Induced Complexation of Soy Protein and Rutin: Improved Interfacial Properties and Emulsion Stability.

    Science.gov (United States)

    Chen, Xiao-Wei; Wang, Jin-Mei; Yang, Xiao-Quan; Qi, Jun-Ru; Hou, Jun-Jie

    2016-09-01

    Rutin is a common dietary flavonoid with important antioxidant and pharmacological activities. However, its application in the food industry is limited mainly because of its poor water solubility. The subcritical water (SW) treatment provides an efficient technique to solubilize and achieve the enrichment of rutin in soy protein isolate (SPI) by inducing their complexation. The physicochemical, interfacial, and emulsifying properties of the complex were investigated and compared to the mixtures. SW treatment had much enhanced rutin-combined capacity of SPI than that of conventional method, ascribing to the well-contacted for higher water solubility of rutin with stronger collision-induced hydrophobic interactions. Compared to the mixtures of rutin with proteins, the complex exhibited an excellent surface activity and improved the physical and oxidative stability of its stabilized emulsions. This improving effect could be attributed to the targeted accumulation of rutin at the oil-water interface accompanied by the adsorption of SPI resulting in the thicker interfacial layer, as evidenced by higher interfacial protein and rutin concentrations. This study provides a novel strategy for the design and enrichment of nanovehicle providing water-insoluble hydrophobic polyphenols for interfacial delivery in food emulsified systems.

  20. Dendritic-tumor fusion cells derived heat shock protein70-peptide complex has enhanced immunogenicity.

    Science.gov (United States)

    Zhang, Yunfei; Zhang, Yong; Chen, Jun; Liu, Yunyan; Luo, Wen

    2015-01-01

    Tumor-derived heat shock protein70-peptide complexes (HSP70.PC-Tu) have shown great promise in tumor immunotherapy due to numerous advantages. However, large-scale phase III clinical trials showed that the limited immunogenicity remained to be enhanced. In previous research, we demonstrated that heat shock protein 70-peptide complexes (HSP70.PC-Fc) derived from dendritic cell (DC)-tumor fusions exhibit enhanced immunogenicity compared with HSP70.PCs from tumor cells. However, the DCs used in our previous research were obtained from healthy donors and not from the patient population. In order to promote the clinical application of these complexes, HSP70.PC-Fc was prepared from patient-derived DC fused directly with patient-derived tumor cells in the current study. Our results showed that compared with HSP70.PC-Tu, HSP70.PC-Fc elicited much more powerful immune responses against the tumor from which the HSP70 was derived, including enhanced T cell activation, and CTL responses that were shown to be antigen specific and HLA restricted. Our results further indicated that the enhanced immunogenicity is related to the activation of CD4+ T cells and increased association with other heat shock proteins, such as HSP90. Therefore, the current study confirms the enhanced immunogenicity of HSP70.PC derived from DC-tumor fusions and may provide direct evidence promoting their future clinical use.

  1. Multiplex matrix network analysis of protein complexes in the human TCR signalosome.

    Science.gov (United States)

    Smith, Stephen E P; Neier, Steven C; Reed, Brendan K; Davis, Tessa R; Sinnwell, Jason P; Eckel-Passow, Jeanette E; Sciallis, Gabriel F; Wieland, Carilyn N; Torgerson, Rochelle R; Gil, Diana; Neuhauser, Claudia; Schrum, Adam G

    2016-08-02

    Multiprotein complexes transduce cellular signals through extensive interaction networks, but the ability to analyze these networks in cells from small clinical biopsies is limited. To address this, we applied an adaptable multiplex matrix system to physiologically relevant signaling protein complexes isolated from a cell line or from human patient samples. Focusing on the proximal T cell receptor (TCR) signalosome, we assessed 210 pairs of PiSCES (proteins in shared complexes detected by exposed surface epitopes). Upon stimulation of Jurkat cells with superantigen-loaded antigen-presenting cells, this system produced high-dimensional data that enabled visualization of network activity. A comprehensive analysis platform generated PiSCES biosignatures by applying unsupervised hierarchical clustering, principal component analysis, an adaptive nonparametric with empirical cutoff analysis, and weighted correlation network analysis. We generated PiSCES biosignatures from 4-mm skin punch biopsies from control patients or patients with the autoimmune skin disease alopecia areata. This analysis distinguished disease patients from the controls, detected enhanced basal TCR signaling in the autoimmune patients, and identified a potential signaling network signature that may be indicative of disease. Thus, generation of PiSCES biosignatures represents an approach that can provide information about the activity of protein signaling networks in samples including low-abundance primary cells from clinical biopsies.

  2. Multiplex matrix network analysis of protein complexes in the human TCR signalosome.

    Science.gov (United States)

    Smith, Stephen E P; Neier, Steven C; Reed, Brendan K; Davis, Tessa R; Sinnwell, Jason P; Eckel-Passow, Jeanette E; Sciallis, Gabriel F; Wieland, Carilyn N; Torgerson, Rochelle R; Gil, Diana; Neuhauser, Claudia; Schrum, Adam G

    2016-01-01

    Multiprotein complexes transduce cellular signals through extensive interaction networks, but the ability to analyze these networks in cells from small clinical biopsies is limited. To address this, we applied an adaptable multiplex matrix system to physiologically relevant signaling protein complexes isolated from a cell line or from human patient samples. Focusing on the proximal T cell receptor (TCR) signalosome, we assessed 210 pairs of PiSCES (proteins in shared complexes detected by exposed surface epitopes). Upon stimulation of Jurkat cells with superantigen-loaded antigen-presenting cells, this system produced high-dimensional data that enabled visualization of network activity. A comprehensive analysis platform generated PiSCES biosignatures by applying unsupervised hierarchical clustering, principal component analysis, an adaptive nonparametric with empirical cutoff analysis, and weighted correlation network analysis. We generated PiSCES biosignatures from 4-mm skin punch biopsies from control patients or patients with the autoimmune skin disease alopecia areata. This analysis distinguished disease patients from the controls, detected enhanced basal TCR signaling in the autoimmune patients, and identified a potential signaling network signature that may be indicative of disease. Thus, generation of PiSCES biosignatures represents an approach that can provide information about the activity of protein signaling networks in samples including low-abundance primary cells from clinical biopsies. PMID:27485017

  3. A novel method for preparation of HAMLET-like protein complexes.

    Science.gov (United States)

    Permyakov, Sergei E; Knyazeva, Ekaterina L; Leonteva, Marina V; Fadeev, Roman S; Chekanov, Aleksei V; Zhadan, Andrei P; Håkansson, Anders P; Akatov, Vladimir S; Permyakov, Eugene A

    2011-09-01

    Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca(2+) transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties.

  4. Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes.

    Science.gov (United States)

    Zhou, Wu; Li, Xiaobo; Premont, Richard T

    2016-05-15

    The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT-PIX complex in health and disease. PMID:27182061

  5. RPGR-containing protein complexes in syndromic and non-syndromic retinal degeneration due to ciliary dysfunction

    Indian Academy of Sciences (India)

    Carlos A. Murga-Zamalloa; Anand Swaroop; Hemant Khanna

    2009-12-01

    Dysfunction of primary cilia due to mutations in cilia-centrosomal proteins is associated with pleiotropic disorders. The primary (or sensory) cilium of photoreceptors mediates polarized trafficking of proteins for efficient phototransduction. Retinitis pigmentosa GTPase regulator (RPGR) is a cilia-centrosomal protein mutated in >70% of X-linked RP cases and 10%–20% of simplex RP males. Accumulating evidence indicates that RPGR may facilitate the orchestration of multiple ciliary protein complexes. Disruption of these complexes due to mutations in component proteins is an underlying cause of associated photoreceptor degeneration. Here, we highlight the recent developments in understanding the mechanism of cilia-dependent photoreceptor degeneration due to mutations in RPGR and RPGR-interacting proteins in severe genetic diseases, including retinitis pigmentosa, Leber congenital amaurosis (LCA), Joubert syndrome, and Senior–Loken syndrome, and explore the physiological relevance of photoreceptor ciliary protein complexes.

  6. Data on structural transitions in domains of hordeivirus TGB1 protein forming ribonucleoprotein complex.

    Science.gov (United States)

    Makarov, Valentin V; Makarova, Svetlana S; Kalinina, Natalia O

    2016-09-01

    This data article is related to the research article entitled "in vitro properties of hordeivirus TGB1 protein forming ribonucleoprotein complexes" (Makarov et al., 2015 [1]), demonstrating that upon incubation with viral RNA the poa semilatent hordeivirus (PSLV) TGB1 protein (the movement 63 K protein encoded by the first gene of the triple gene block) in vitro forms RNP structures resembling filamentous virus-like particles and its internal domain (ID) performs a major structural role in this process. This article reports the additional results on the structural lability of ID and the structural transitions in the C-terminal NTPase/helicase domain (HELD) induced by interaction with tRNA and phosphorylation. PMID:27331098

  7. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    International Nuclear Information System (INIS)

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrPC. In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrPC at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  8. Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex.

    Science.gov (United States)

    Vovk, Andrei; Gu, Chad; Opferman, Michael G; Kapinos, Larisa E; Lim, Roderick Yh; Coalson, Rob D; Jasnow, David; Zilman, Anton

    2016-01-01

    Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function. PMID:27198189

  9. Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17

    OpenAIRE

    Howard, Paul W.; Jue, Shall F.; Maurer, Richard A.

    2013-01-01

    Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known...

  10. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. [Dynamic model of protein behavior in water. Possible mechanism of association and dissociation of specific complexes].

    Science.gov (United States)

    Kiaiviariainen, A I

    1979-01-01

    The proposed mechanism of association and dissociation of specific protein complex with ligands is based on the assumption that thermal fluctuations in the non-polar cavities of protein are interrelated due to the co-operative properties of macromolecules. The non-polar cavities, representing the active centres and clefts in the globule jump from the "open" state to the "closed" one with a certain amount of water being removed to the external medium. It is assumed that the association of ligand with the active centre is accompanied by stabilization of the chest state and destibilization of the open one, dissociation being a reverse process. A rapid change in the state of the active centre under the influence of ligand induces a slow relaxation process resulting in similar alterations in the state of the auxiliary non-polar cavities of protein. As a result, the binding constant of ligand increases, while the free energy of protein decreases. Expressions have been obtained which relate the rate constants of association, dissociation and the binding constant to the rate-constant of the nonpolar cavities transitions from the closed state to the open one, and vice versa. They gave a possibility for a qualitative interpretation of the effect of some specific and non-specific agents upon the stability of immunoglobulins, on the influence of salts upon the association and dissociation of antibody-antigene complexes and on the increase in the constant of binding between antibodies and haptens in the course of prolonged immunisation.

  12. Distinct configurations of protein complexes and biochemical pathways revealed by epistatic interaction network motifs

    LENUS (Irish Health Repository)

    Casey, Fergal

    2011-08-22

    Abstract Background Gene and protein interactions are commonly represented as networks, with the genes or proteins comprising the nodes and the relationship between them as edges. Motifs, or small local configurations of edges and nodes that arise repeatedly, can be used to simplify the interpretation of networks. Results We examined triplet motifs in a network of quantitative epistatic genetic relationships, and found a non-random distribution of particular motif classes. Individual motif classes were found to be associated with different functional properties, suggestive of an underlying biological significance. These associations were apparent not only for motif classes, but for individual positions within the motifs. As expected, NNN (all negative) motifs were strongly associated with previously reported genetic (i.e. synthetic lethal) interactions, while PPP (all positive) motifs were associated with protein complexes. The two other motif classes (NNP: a positive interaction spanned by two negative interactions, and NPP: a negative spanned by two positives) showed very distinct functional associations, with physical interactions dominating for the former but alternative enrichments, typical of biochemical pathways, dominating for the latter. Conclusion We present a model showing how NNP motifs can be used to recognize supportive relationships between protein complexes, while NPP motifs often identify opposing or regulatory behaviour between a gene and an associated pathway. The ability to use motifs to point toward underlying biological organizational themes is likely to be increasingly important as more extensive epistasis mapping projects in higher organisms begin.

  13. Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

    Directory of Open Access Journals (Sweden)

    Christina Looße

    2015-06-01

    Full Text Available Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4 comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m. deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.

  14. Binding of the human papillomavirus E1 origin-recognition protein is regulated through complex formation with the E2 enhancer-binding protein.

    OpenAIRE

    Frattini, M G; Laimins, L A

    1994-01-01

    The papillomavirus E1 and E2 proteins form heteromeric complexes and individually bind specific sequences within the viral origin of replication. The mechanism by which these proteins are recruited to the origin and the role of the E1/E2 complex in replication remain undefined. To examine the interplay of these replication proteins, we have analyzed the binding of human papillomavirus (HPV) type 31b E1 and E2 proteins to the origin of replication. Binding of E1 to the origin was increased by ...

  15. Microencapsulation of chia seed oil using chia seed protein isolate-chia seed gum complex coacervates.

    Science.gov (United States)

    Timilsena, Yakindra Prasad; Adhikari, Raju; Barrow, Colin J; Adhikari, Benu

    2016-10-01

    Chia seed oil (CSO) microcapsules were produced by using chia seed protein isolate (CPI)-chia seed gum (CSG) complex coacervates aiming to enhance the oxidative stability of CSO. The effect of wall material composition, core-to-wall ratio and method of drying on the microencapsulation efficiency (MEE) and oxidative stability (OS) was studied The microcapsules produced using CPI-CSG complex coacervates as wall material had higher MEE at equivalent payload, lower surface oil and higher OS compared to the microcapsules produced by using CSG and CPI individually. CSO microcapsules produced by using CSG as wall material had lowest MEE (67.3%) and oxidative stability index (OSI=6.6h), whereas CPI-CSG complex coacervate microcapsules had the highest MEE (93.9%) and OSI (12.3h). The MEE and OSI of microcapsules produced by using CPI as wall materials were in between those produced by using CSG and CPI-CSG complex coacervates as wall materials. The CSO microcapsules produced by using CPI-CSG complex coacervate as shell matrix at core-to-wall ratio of 1:2 had 6 times longer storage life compared to that of unencapsulated CSO. The peroxide value of CSO microcapsule produced using CPI-CSG complex coacervate as wall material was <10meq O2/kg oil during 30 days of storage.

  16. Conformational fluctuations of a protein-DNA complex and the structure and ordering of water around it

    Science.gov (United States)

    Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy

    2011-12-01

    Protein-DNA binding is an important process responsible for the regulation of genetic activities in living organisms. The most crucial issue in this problem is how the protein recognizes the DNA and identifies its target base sequences. Water molecules present around the protein and DNA are also expected to play an important role in mediating the recognition process and controlling the structure of the complex. We have performed atomistic molecular dynamics simulations of an aqueous solution of the protein-DNA complex formed between the DNA binding domain of human TRF1 protein and a telomeric DNA. The conformational fluctuations of the protein and DNA and the microscopic structure and ordering of water around them in the complex have been explored. In agreement with experimental studies, the calculations reveal conformational immobilization of the terminal segments of the protein on complexation. Importantly, it is discovered that both structural adaptations of the protein and DNA, and the subsequent correlation between them to bind, contribute to the net entropy loss associated with the complex formation. Further, it is found that water molecules around the DNA are more structured with significantly higher density and ordering than that around the protein in the complex.

  17. Computational analysis of the CB1 carboxyl-terminus in the receptor-G protein complex.

    Science.gov (United States)

    Shim, Joong-Youn; Khurana, Leepakshi; Kendall, Debra A

    2016-04-01

    Despite the important role of the carboxyl-terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1-Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449-32465) to incorporate a complete CB1 Ct (Glu416(Ct)-Leu472(Ct)). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1-Gi complex. The resulting models were consistent with the NMR-determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site-directed mutagenesis studies of Glu416(Ct), Asp423(Ct), Asp428(Ct), and Arg444(Ct) of CB1 Ct suggested that the CB1 Ct can influence receptor-G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein. PMID:26994549

  18. Computational analysis of the CB1 carboxyl-terminus in the receptor-G protein complex.

    Science.gov (United States)

    Shim, Joong-Youn; Khurana, Leepakshi; Kendall, Debra A

    2016-04-01

    Despite the important role of the carboxyl-terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1-Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449-32465) to incorporate a complete CB1 Ct (Glu416(Ct)-Leu472(Ct)). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1-Gi complex. The resulting models were consistent with the NMR-determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site-directed mutagenesis studies of Glu416(Ct), Asp423(Ct), Asp428(Ct), and Arg444(Ct) of CB1 Ct suggested that the CB1 Ct can influence receptor-G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein.

  19. Evolution of light-harvesting complex proteins from Chl c-containing algae

    Directory of Open Access Journals (Sweden)

    Puerta M Virginia

    2011-04-01

    Full Text Available Abstract Background Light harvesting complex (LHC proteins function in photosynthesis by binding chlorophyll (Chl and carotenoid molecules that absorb light and transfer the energy to the reaction center Chl of the photosystem. Most research has focused on LHCs of plants and chlorophytes that bind Chl a and b and extensive work on these proteins has uncovered a diversity of biochemical functions, expression patterns and amino acid sequences. We focus here on a less-studied family of LHCs that typically bind Chl a and c, and that are widely distributed in Chl c-containing and other algae. Previous phylogenetic analyses of these proteins suggested that individual algal lineages possess proteins from one or two subfamilies, and that most subfamilies are characteristic of a particular algal lineage, but genome-scale datasets had revealed that some species have multiple different forms of the gene. Such observations also suggested that there might have been an important influence of endosymbiosis in the evolution of LHCs. Results We reconstruct a phylogeny of LHCs from Chl c-containing algae and related lineages using data from recent sequencing projects to give ~10-fold larger taxon sampling than previous studies. The phylogeny indicates that individual taxa possess proteins from multiple LHC subfamilies and that several LHC subfamilies are found in distantly related algal lineages. This phylogenetic pattern implies functional differentiation of the gene families, a hypothesis that is consistent with data on gene expression, carotenoid binding and physical associations with other LHCs. In all probability LHCs have undergone a complex history of evolution of function, gene transfer, and lineage-specific diversification. Conclusion The analysis provides a strikingly different picture of LHC diversity than previous analyses of LHC evolution. Individual algal lineages possess proteins from multiple LHC subfamilies. Evolutionary relationships showed

  20. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins. PMID:24297163

  1. Demonstrating an Order-of-Magnitude Sampling Enhancement in Molecular Dynamics Simulations of Complex Protein Systems.

    Science.gov (United States)

    Pan, Albert C; Weinreich, Thomas M; Piana, Stefano; Shaw, David E

    2016-03-01

    Molecular dynamics (MD) simulations can describe protein motions in atomic detail, but transitions between protein conformational states sometimes take place on time scales that are infeasible or very expensive to reach by direct simulation. Enhanced sampling methods, the aim of which is to increase the sampling efficiency of MD simulations, have thus been extensively employed. The effectiveness of such methods when applied to complex biological systems like proteins, however, has been difficult to establish because even enhanced sampling simulations of such systems do not typically reach time scales at which convergence is extensive enough to reliably quantify sampling efficiency. Here, we obtain sufficiently converged simulations of three proteins to evaluate the performance of simulated tempering, a member of a widely used class of enhanced sampling methods that use elevated temperature to accelerate sampling. Simulated tempering simulations with individual lengths of up to 100 μs were compared to (previously published) conventional MD simulations with individual lengths of up to 1 ms. With two proteins, BPTI and ubiquitin, we evaluated the efficiency of sampling of conformational states near the native state, and for the third, the villin headpiece, we examined the rate of folding and unfolding. Our comparisons demonstrate that simulated tempering can consistently achieve a substantial sampling speedup of an order of magnitude or more relative to conventional MD.

  2. Bacterial conjugation protein MobA mediates integration of complex DNA structures into plant cells.

    Science.gov (United States)

    Bravo-Angel, A M; Gloeckler, V; Hohn, B; Tinland, B

    1999-09-01

    Agrobacterium tumefaciens transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by Agrobacterium to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5' end. Isolation and analysis of the most conserved 5' ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins. PMID:10482518

  3. Myc protein is stabilized by suppression of a novel E3 ligase complex in cancer cells

    Science.gov (United States)

    Choi, Seung H.; Wright, Jason B.; Gerber, Scott A.; Cole, Michael D.

    2010-01-01

    Rapid Myc protein turnover is critical for maintaining basal levels of Myc activity in normal cells and a prompt response to changing growth signals. We characterize a new Myc-interacting factor, TRPC4AP (transient receptor potential cation channel, subfamily C, member 4-associated protein)/TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein), which is the receptor for a DDB1 (damage-specific DNA-binding protein 1)–CUL4 (Cullin 4) E3 ligase complex for selective Myc degradation through the proteasome. TRPC4AP/TRUSS binds specifically to the Myc C terminus and promotes its ubiquitination and destruction through the recognition of evolutionarily conserved domains in the Myc N terminus. TRPC4AP/TRUSS suppresses Myc-mediated transactivation and transformation in a dose-dependent manner. Finally, we found that TRPC4AP/TRUSS expression is strongly down-regulated in most cancer cell lines, leading to Myc protein stabilization. These studies identify a novel pathway targeting Myc degradation that is suppressed in cancer cells. PMID:20551172

  4. Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies

    Energy Technology Data Exchange (ETDEWEB)

    Cauchi, Ruben J.; Sanchez-Pulido, Luis [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX (United Kingdom); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX (United Kingdom)

    2010-08-15

    Uridine-rich small nuclear ribonucleoproteins (U snRNPs) play key roles in pre-mRNA processing in the nucleus. The assembly of most U snRNPs takes place in the cytoplasm and is facilitated by the survival motor neuron (SMN) complex. Discrete cytoplasmic RNA granules called U bodies have been proposed to be specific sites for snRNP assembly because they contain U snRNPs and SMN. U bodies invariably associate with P bodies, which are involved in mRNA decay and translational control. However, it remains unknown whether other SMN complex proteins also localise to U bodies. In Drosophila there are four SMN complex proteins, namely SMN, Gemin2/CG10419, Gemin3 and Gemin5/Rigor mortis. Drosophila Gemin3 was originally identified as the Drosophila orthologue of human and yeast Dhh1, a component of P bodies. Through an in silico analysis of the DEAD-box RNA helicases we confirmed that Gemin3 is the bona fide Drosophila orthologue of vertebrate Gemin3 whereas the Drosophila orthologue of Dhh1 is Me31B. We then made use of the Drosophila egg chamber as a model system to study the subcellular distribution of the Gemin proteins as well as Me31B. Our cytological investigations show that Gemin2, Gemin3 and Gemin5 colocalise with SMN in U bodies. Although they are excluded from P bodies, as components of U bodies, Gemin2, Gemin3 and Gemin5 are consistently found associated with P bodies, wherein Me31B resides. In addition to a role in snRNP biogenesis, SMN complexes residing in U bodies may also be involved in mRNP assembly and/or transport.

  5. A Protein Data Bank survey reveals shortening of intermolecular hydrogen bonds in ligand-protein complexes when a halogenated ligand is an H-bond donor.

    Directory of Open Access Journals (Sweden)

    Jarosław Poznański

    Full Text Available Halogen bonding in ligand-protein complexes is currently widely exploited, e.g. in drug design or supramolecular chemistry. But little attention has been directed to other effects that may result from replacement of a hydrogen by a strongly electronegative halogen. Analysis of almost 30000 hydrogen bonds between protein and ligand demonstrates that the length of a hydrogen bond depends on the type of donor-acceptor pair. Interestingly, lengths of hydrogen bonds between a protein and a halogenated ligand are visibly shorter than those estimated for the same family of proteins in complexes with non-halogenated ligands. Taking into account the effect of halogenation on hydrogen bonding is thus important when evaluating structural and/or energetic parameters of ligand-protein complexes. All these observations are consistent with the concept that halogenation increases the acidity of the proximal amino/imino/hydroxyl groups and thus makes them better, i.e. stronger, H-bond donors.

  6. Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

    Energy Technology Data Exchange (ETDEWEB)

    Inui, Ken [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Sagane, Yoshimasa [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Miyata, Keita [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Miyashita, Shin-Ichiro [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Suzuki, Tomonori [Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Shikamori, Yasuyuki [Agilent Technologies International Japan, Ltd. Takaura-cho 9-1, Hachioji-shi, Tokyo 192-0033 (Japan); Ohyama, Tohru; Niwa, Koichi [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Watanabe, Toshihiro, E-mail: t-watana@bioindustry.nodai.ac.jp [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer BoNT and NTNHA proteins share a similar protein architecture. Black-Right-Pointing-Pointer NTNHA and BoNT were both identified as zinc-binding proteins. Black-Right-Pointing-Pointer NTNHA does not have a classical HEXXH zinc-coordinating motif similar to that found in all serotypes of BoNT. Black-Right-Pointing-Pointer Homology modeling implied probable key residues involved in zinc coordination. -- Abstract: Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X{sub 35}-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.

  7. Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

    International Nuclear Information System (INIS)

    Highlights: ► BoNT and NTNHA proteins share a similar protein architecture. ► NTNHA and BoNT were both identified as zinc-binding proteins. ► NTNHA does not have a classical HEXXH zinc-coordinating motif similar to that found in all serotypes of BoNT. ► Homology modeling implied probable key residues involved in zinc coordination. -- Abstract: Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X35-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.

  8. A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

    Institute of Scientific and Technical Information of China (English)

    Waranya Imprasittichai; Sittiruk Roytrakul; Sudaratana R Krungkrai; Jerapan Krungkrai

    2014-01-01

    Objective:To investigate the multienzyme complex formation of human malaria parasite Plasmodium falciparum(P. falciparum) orotate phosphoribosyltransferase(OPRT) and orotidine 5'-monophosphate decarboxylase(OMPDC), the fifth and sixth enzyme of the de novo pyrimidine biosynthetic pathway.Previously, we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)2(OMPDC)2 complex containing two subunits each ofOPRT andOMPDC, and that the complex have catalytic kinetic advantages over the monofunctional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctional chemical cross-linker, liquid chromatography-mass spectrometric analysis and homology modeling.Results:The unique insertions of low complexity region at the α2 and α5 helices of the parasiteOMPDC, characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms, was located on theOPRT-OMPDC interface.The structural models for the protein-protein interaction of the heterotetrameric(OPRT)2(OMPDC)2 multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling, it is surmised that the human malaria parasite low complexity region is responsible for theOPRT-OMPDC interaction.The structural complex of the parasite enzymes, thus, represents an efficient functional kinetic advantage, which in line with co-localization principles of evolutional origin, and allosteric control in protein-protein-interactions.

  9. In various protein complexes, disordered protomers have large per-residue surface areas and area of protein-, DNA- and RNA-binding interfaces.

    Science.gov (United States)

    Wu, Zhonghua; Hu, Gang; Yang, Jianyi; Peng, Zhenling; Uversky, Vladimir N; Kurgan, Lukasz

    2015-09-14

    We provide first large scale analysis of the peculiarities of surface areas of 5658 dissimilar (below 50% sequence similarity) proteins with known 3D-structures that bind to proteins, DNA or RNAs. We show here that area of the protein surface is highly correlated with the protein length. The size of the interface surface is only modestly correlated with the protein size, except for RNA-binding proteins where larger proteins are characterized by larger interfaces. Disordered proteins with disordered interfaces are characterized by significantly larger per-residue areas of their surfaces and interfaces when compared to the structured proteins. These result are applicable for proteins involved in interaction with DNA, RNA, and proteins and suggest that disordered proteins and binding regions are less compact and more likely to assume extended shape. We demonstrate that disordered protein binding residues in the interfaces of disordered proteins drive the increase in the per residue area of these interfaces. Our results can be used to predict in silico whether a given protomer from the DNA, RNA or protein complex is likely to be disordered in its unbound form.

  10. Relationship between residual feed intake and lymphocyte mitochondrial complex protein concentration and ratio in crossbred steers.

    Science.gov (United States)

    Davis, M P; Brooks, M A; Kerley, M S

    2016-04-01

    Rate of oxygen uptake by muscle mitochondria and respiratory chain protein concentrations differed between high- and low-residual feed intake (RFI) animals. The hypothesis of this research was that complex I (CI), II (CII), and III (CIII) mitochondria protein concentrations in lymphocyte (blood) mitochondria were related to the RFI phenotype of beef steers. Daily feed intake (ADFI) was individually recorded for 92 Hereford-crossbreed steers over 63 d using GrowSafe individual feed intake system. Predicted ADFI was calculated as the regression of ADFI on ADG and midtest BW. Difference between ADFI and predicted ADFI was RFI. Lymphocytes were isolated from low-RFI (-1.32 ± 0.11 kg/d; = 10) and high-RFI (1.34 ± 0.18 kg/d; = 8) steers. Immunocapture of CI, CII, and CIII proteins from the lymphocyte was done using MitoProfile CI, CII, and CIII immunocapture kits (MitoSciences Inc., Eugene, OR). Protein concentrations of CI, CII, and CIII and total protein were quantified using bicinchoninic acid colorimetric procedures. Low-RFI steers consumed 30% less ( = 0.0004) feed and had a 40% improvement ( RFI steers with similar growth ( = 0.78) and weight measurements ( > 0.65). High- and low-RFI steers did not differ in CI ( = 0.22), CII ( = 0.69), and CIII ( = 0.59) protein concentrations. The protein concentration ratios for CI to CII ( = 0.03) were 20% higher and the ratios of CI to CIII ( = 0.01) were 30% higher, but the ratios of CII to CIII ( = 0.89) did not differ when comparing low-RFI steers with high-RFI steers. The similar magnitude difference in feed intake, feed efficiency measurements, and CI-to-CIII ratio between RFI phenotypes provides a plausible explanation for differences between the phenotypes. We also concluded that mitochondria isolated from lymphocytes could be used to study respiratory chain differences among differing RFI phenotypes. Further research is needed to determine if lymphocyte mitochondrial complex proteins can be used for identification of

  11. Synthesis, characterization, DNA binding, DNA cleavage, protein binding and cytotoxic activities of Ru(II) complexes.

    Science.gov (United States)

    Thota, Sreekanth; Vallala, Srujana; Yerra, Rajeshwar; Rodrigues, Daniel Alencar; Raghavendra, Nulgumnalli Manjunathaiah; Barreiro, Eliezer J

    2016-01-01

    We report on the synthesis of novel Ru(II) compounds (Ru-1 to Ru-8) bearing R-pdc, 4-Cl-pbinh ligands (where R=4-CF3, 4-F, 4-OH pdc=3-phenyl-5-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, pbinh=phenoxybenzylidene isonicotinyl hydrazides) and their in vitro antitumor activity toward the cell lines murine leukemia L1210, human lymphocyte CEM, human epithelial cervical carcinoma HeLa, BEL-7402 and Molt4/C8. Some of the complexes exhibited more potent antiproliferative activity against cell lines than the standard drug cisplatin. Ruthenium complex Ru-2 displayed potent cytotoxicity with than that of cisplatin. DNA-binding, DNA cleavage and protein binding properties of ruthenium complexes with these ligands are reported. Interactions of these ruthenium complexes with DNA revealed an intercalative mode of binding between them. Synchronous fluorescence spectra proved that the interaction of ruthenium complexes with bovine serum albumin (BSA) resulted in a conformational change of the latter.

  12. “Non-Toxic” Proteins of the Botulinum Toxin Complex Exert In-vivo Toxicity

    Science.gov (United States)

    Miyashita, Shin-Ichiro; Sagane, Yoshimasa; Suzuki, Tomonori; Matsumoto, Takashi; Niwa, Koichi; Watanabe, Toshihiro

    2016-01-01

    The botulinum neurotoxin (BoNT) causes muscle paralysis and is the most potent toxin in nature. BoNT is associated with a complex of auxiliary “Non-Toxic” proteins, which constitute a large-sized toxin complex (L-TC). However, here we report that the “Non-Toxic” complex of serotype D botulinum L-TC, when administered to rats, exerts in-vivo toxicity on small-intestinal villi. Moreover, Serotype C and D of the “Non-Toxic” complex, but not BoNT, induced vacuole-formation in a rat intestinal epithelial cell line (IEC-6), resulting in cell death. Our results suggest that the vacuole was formed in a manner distinct from the mechanism by which Helicobacter pylori vacuolating toxin (VacA) and Vibrio cholerae haemolysin induce vacuolation. We therefore hypothesise that the serotype C and D botulinum toxin complex is a functional hybrid of the neurotoxin and vacuolating toxin (VT) which arose from horizontal gene transfer from an ancestral BoNT-producing bacterium to a hypothetical VT-producing bacterium. PMID:27507612

  13. "Non-Toxic" Proteins of the Botulinum Toxin Complex Exert In-vivo Toxicity.

    Science.gov (United States)

    Miyashita, Shin-Ichiro; Sagane, Yoshimasa; Suzuki, Tomonori; Matsumoto, Takashi; Niwa, Koichi; Watanabe, Toshihiro

    2016-01-01

    The botulinum neurotoxin (BoNT) causes muscle paralysis and is the most potent toxin in nature. BoNT is associated with a complex of auxiliary "Non-Toxic" proteins, which constitute a large-sized toxin complex (L-TC). However, here we report that the "Non-Toxic" complex of serotype D botulinum L-TC, when administered to rats, exerts in-vivo toxicity on small-intestinal villi. Moreover, Serotype C and D of the "Non-Toxic" complex, but not BoNT, induced vacuole-formation in a rat intestinal epithelial cell line (IEC-6), resulting in cell death. Our results suggest that the vacuole was formed in a manner distinct from the mechanism by which Helicobacter pylori vacuolating toxin (VacA) and Vibrio cholerae haemolysin induce vacuolation. We therefore hypothesise that the serotype C and D botulinum toxin complex is a functional hybrid of the neurotoxin and vacuolating toxin (VT) which arose from horizontal gene transfer from an ancestral BoNT-producing bacterium to a hypothetical VT-producing bacterium. PMID:27507612

  14. The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation

    DEFF Research Database (Denmark)

    Müller, Oliver; Bayer, Martin J; Peters, Christopher;

    2002-01-01

    The fusion of cellular membranes comprises several steps; membrane attachment requires priming of SNAREs and tethering factors by Sec18p/NSF (N-ethylmaleimide sensitive factor) and LMA1. This leads to trans-SNARE pairing, i.e. formation of SNARE complexes between apposed membranes. The yeast...... vacuole system has revealed two subsequent molecular events: trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release of LMA1 from the membrane. We have now identified a hetero-oligomeric membrane integral complex of vacuolar transporter chaperone (Vtc) proteins integrating these events....... The Vtc complex associates with the R-SNARE Nyv1p and with V(0). Subunits Vtc1p and Vtc4p control the initial steps of fusion. They are required for Sec18p/NSF activity in SNARE priming, membrane binding of LMA1 and V(0) trans-complex formation. In contrast, subunit Vtc3p is required for the latest step...

  15. Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

    DEFF Research Database (Denmark)

    Mygind, Per H; Christiansen, Gunna; Roepstorff, P;

    2000-01-01

    The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By....... By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH....

  16. Consistent two-dimensional visualization of protein-ligand complex series

    Directory of Open Access Journals (Sweden)

    Stierand Katrin

    2011-06-01

    Full Text Available Abstract Background The comparative two-dimensional graphical representation of protein-ligand complex series featuring different ligands bound to the same active site offers a quick insight in their binding mode differences. In comparison to arbitrary orientations of the residue molecules in the individual complex depictions a consistent placement improves the legibility and comparability within the series. The automatic generation of such consistent layouts offers the possibility to apply it to large data sets originating from computer-aided drug design methods. Results We developed a new approach, which automatically generates a consistent layout of interacting residues for a given series of complexes. Based on the structural three-dimensional input information, a global two-dimensional layout for all residues of the complex ensemble is computed. The algorithm incorporates the three-dimensional adjacencies of the active site residues in order to find an universally valid circular arrangement of the residues around the ligand. Subsequent to a two-dimensional ligand superimposition step, a global placement for each residue is derived from the set of already placed ligands. The method generates high-quality layouts, showing mostly overlap-free solutions with molecules which are displayed as structure diagrams providing interaction information in atomic detail. Application examples document an improved legibility compared to series of diagrams whose layouts are calculated independently from each other. Conclusions The presented method extends the field of complex series visualizations. A series of molecules binding to the same protein active site is drawn in a graphically consistent way. Compared to existing approaches these drawings substantially simplify the visual analysis of large compound series.

  17. Detection of two isomeric binding configurations in a protein-aptamer complex with a biological nanopore.

    Science.gov (United States)

    Van Meervelt, Veerle; Soskine, Misha; Maglia, Giovanni

    2014-12-23

    Protein-DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we showed that folded proteins can be internalized inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalization of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores can be used to probe conformational heterogeneity in protein:DNA interactions. PMID:25493908

  18. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  19. Predicting protein structural classes based on complex networks and recurrence analysis.

    Science.gov (United States)

    Olyaee, Mohammad H; Yaghoubi, Ali; Yaghoobi, Mahdi

    2016-09-01

    Protein sequences are divided into four structural classes. The determination of class is a challenging and beneficial task in the bioinformatics field. Several methods have been proposed to this end, but most utilize too many features and produce unsuitable results. In the present, features are extracted based on the predicted secondary structures. At first, predicted secondary structure sequences are mapped into two time series by the chaos game representation. Then, a recurrence matrix is calculated from each of the time series. The recurrence matrix is identified with the adjacency matrix of a complex network and measures are applied for the characterization of complex networks to these recurrence matrixes. For a given protein sequence, a total of 24 characteristic features can be calculated and these are fed into Fisher's discriminated analysis algorithm for classification. To examine the proposed method, two widely used low similarity benchmark datasets design and test its performance. A comparison with the results of existing methods shows that the current study's approach provides a satisfactory performance for protein structural class prediction. PMID:27320678

  20. Ion mobility mass spectrometry of peptide, protein, and protein complex ions using a radio-frequency confining drift cell.

    Science.gov (United States)

    Allen, Samuel J; Giles, Kevin; Gilbert, Tony; Bush, Matthew F

    2016-02-01

    Ion mobility mass spectrometry experiments enable the characterization of mass, assembly, and shape of biological molecules and assemblies. Here, a new radio-frequency confining drift cell is characterized and used to measure the mobilities of peptide, protein, and protein complex ions. The new drift cell replaced the traveling-wave ion mobility cell in a Waters Synapt G2 HDMS. Methods for operating the drift cell and determining collision cross section values using this experimental set up are presented within the context of the original instrument control software. Collision cross sections for 349 cations and anions are reported, 155 of which are for ions that have not been characterized previously using ion mobility. The values for the remaining ions are similar to those determined using a previous radio-frequency confining drift cell and drift tubes without radial confinement. Using this device under 2 Torr of helium gas and an optimized drift voltage, denatured and native-like ions exhibited average apparent resolving powers of 14.2 and 16.5, respectively. For ions with high mobility, which are also low in mass, the apparent resolving power is limited by contributions from ion gating. In contrast, the arrival-time distributions of low-mobility, native-like ions are not well explained using only contributions from ion gating and diffusion. For those species, the widths of arrival-time distributions are most consistent with the presence of multiple structures in the gas phase. PMID:26739109

  1. Ion mobility mass spectrometry of peptide, protein, and protein complex ions using a radio-frequency confining drift cell.

    Science.gov (United States)

    Allen, Samuel J; Giles, Kevin; Gilbert, Tony; Bush, Matthew F

    2016-02-01

    Ion mobility mass spectrometry experiments enable the characterization of mass, assembly, and shape of biological molecules and assemblies. Here, a new radio-frequency confining drift cell is characterized and used to measure the mobilities of peptide, protein, and protein complex ions. The new drift cell replaced the traveling-wave ion mobility cell in a Waters Synapt G2 HDMS. Methods for operating the drift cell and determining collision cross section values using this experimental set up are presented within the context of the original instrument control software. Collision cross sections for 349 cations and anions are reported, 155 of which are for ions that have not been characterized previously using ion mobility. The values for the remaining ions are similar to those determined using a previous radio-frequency confining drift cell and drift tubes without radial confinement. Using this device under 2 Torr of helium gas and an optimized drift voltage, denatured and native-like ions exhibited average apparent resolving powers of 14.2 and 16.5, respectively. For ions with high mobility, which are also low in mass, the apparent resolving power is limited by contributions from ion gating. In contrast, the arrival-time distributions of low-mobility, native-like ions are not well explained using only contributions from ion gating and diffusion. For those species, the widths of arrival-time distributions are most consistent with the presence of multiple structures in the gas phase.

  2. Slow formation of stable complexes during coincubation of a minimal rRNA and ribosomal protein S4

    OpenAIRE

    Mayerle, Megan; Bellur, Deepti L.; Woodson, Sarah A.

    2011-01-01

    Ribosomal protein S4 binds and stabilizes a five-helix junction in the 5’ domain of the 16S rRNA, and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and the five-helix junction reorganize their structures. We show that labile S4 complexes rearrange to stable complexes within a few minutes at 42°C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stab...

  3. Effect of apolipoprotein a-I complex with tetrahydrocortisone on protein biosynthesis and glucose absorption by rat hepatocytes.

    Science.gov (United States)

    Sumenkova, D V; Knyazev, R A; Guschya, R S; Polyakov, L M; Panin, L E

    2009-08-01

    We studied the effect of apolipoprotein A-I-tetrahydrocortisone complex on (14)C glucose absorption and lactate accumulation and on the rate of protein biosynthesis in isolated rat hepatocytes. The presence of apolipoprotein A-I-tetrahydrocortisone complex in the incubation medium increased absorption of labeled glucose by hepatocytes by 52%, while lactate content in the conditioning medium increased 4-fold. The rate of protein biosynthesis increased by 80% in comparison with control cells. It is hypothesized that the increase in protein biosynthesis rate in hepatocytes under the effect of apolipoprotein A-I-tetrahydrocortisone complex is due to stimulation of energy metabolism, specifically, of its glycolytic component. PMID:20027330

  4. The structure of a ribosomal protein S8/spc operon mRNA complex.

    Science.gov (United States)

    Merianos, Helen J; Wang, Jimin; Moore, Peter B

    2004-06-01

    In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.

  5. Anti-inflammatory activity of copper-protein complexes with reference to rheumatoid arthritis

    International Nuclear Information System (INIS)

    Work was carried out on the serum of 100 healthy subjects and hundred Rheumatoid Arthritus patients by electrophoresis and atomic absorption model Hitachi-180-80. The different proteins fractions separated by electrophoresis were subjected for estimation of Cu-Concentration. It was found that only 5% copper is present in albumin which remains constant in normal and Rheumatoid Arthritus patients. The other important group of proteins were a2-globulins, in which Cu concentration was found high in Rheumatoid Arthritus patients as compared to normal subject. There was no copper detected in beta and gamma globulins. The study can be used for diagnostic purposes. Copper complexes of alpha 2-Globulins were found anti inflammatory in nature and this quality of self defense can bring revolution in the history of all infectious diseases including Rheumatoid Arthritus simply by separating the copper complexes of alpha 2-globulins (most probably ceruloplasmin) from the blood of donors and injecting into the body of patients. It is possible by this method that the disease-Rheumatoid Arthritus may be eradicated completely. These complexes have also shown antibiotic activity in gynae patients. (author)

  6. The promyelocytic leukemia gene product (PML) forms stable complexes with the retinoblastoma protein

    DEFF Research Database (Denmark)

    Alcalay, M; Tomassoni, L; Colombo, E;

    1998-01-01

    by the expression of PML-RAR alpha. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RAR alpha expression. Both PML and PML-RAR alpha form complexes with the nonphosphorylated form of pRB in vivo......, and they interact with the pocket region of pRB. The regions of PML and PML-RAR alpha involved in pRB binding differ; in fact, the B boxes and the C-terminal region of PML, the latter of which is not present in PML-RAR alpha, are essential for the formation of stable complexes with pRB. Functionally, PML abolishes...... activation of glucocorticoid receptor-regulated transcription by pRB, whereas PML-RAR alpha further increases it. Our results suggest that PML may be part of transcription-regulatory complexes and that the oncogenic potential of the PML-RAR alpha protein may derive from the alteration of PML...

  7. CONTENTS OF SERUM MYELIN BASIC PROTEIN-IGG ANTIBODIES COMPLEXES IN NORMAL PREGNANCY AND GESTOSIS

    Directory of Open Access Journals (Sweden)

    V. G. Levchenko

    2010-01-01

    Full Text Available Serum levels of myelin basic protein (MBP-bound immune complexes were studied in blood sera from women with gestosis, as compared with those in normal pregnancy and non-pregnant woman. The amounts of IgG-MBP complex in blood serum were determined by enzyme immunoassay using isolated anti-МBP-antibodies. The study has shown that about 0.05 mcg of IgG ml of blood serum are associated with myelin basic protein in unpregnant women or in normal pregnancy. Mild gestosis is accompanied by a 2-3-fold increase in MBP immunocomplex concentrations in blood serum. More severe stages of gestosis are characterized by its further rise, thus achieving maximal values of such MBP immune complexes (0.8 mcg/ml in patients with pre-eclampsia and eclampsia. Their amounts were reduced twice after the periods of eclampsia. Serum levels of MBP-bound IgGs may be used to determine severity of gestosis and to predict a risk of eclampsia in pregnant women.

  8. NMR-guided molecular docking of a protein-peptide complex based on ant colony optimization.

    Science.gov (United States)

    Korb, Oliver; Möller, Heiko M; Exner, Thomas E

    2010-07-01

    Standard docking approaches used for the prediction of protein-ligand complexes in the drug development process have problems identifying the correct binding mode of large flexible ligands. Herein we show how additional experimental data from NMR experiments can be used to predict the binding mode of a mucin 1 (MUC-1) pentapeptide recognized by the breast-cancer-selective monoclonal antibody SM3. Distance constraints derived from trNOE and saturation transfer difference NMR experiments are combined with the docking approach PLANTS. The resulting complex structures show excellent agreement with the NMR data and with a published X-ray crystal structure. The method was then further tested on two complexes in order to demonstrate its more general applicability: T-antigen disaccharide bound to Maclura pomifera agglutinin, and the inhibitor SBi279 bound to S100B protein. Our new approach has the advantages of being fully automatic, rapid, and unbiased; moreover, it is based on relatively easily obtainable experimental data and can greatly increase the reliability of the generated structures. PMID:20486157

  9. A new algorithm for essential proteins identification based on the integration of protein complex co-expression information and edge clustering coefficient.

    Science.gov (United States)

    Luo, Jiawei; Wu, Juan

    2015-01-01

    Essential proteins provide valuable information for the development of biology and medical research from the system level. The accuracy of topological centrality only based methods is deeply affected by noise in the network. Therefore, exploring efficient methods for identifying essential proteins would be of great value. Using biological features to identify essential proteins is efficient in reducing the noise in PPI network. In this paper, based on the consideration that essential proteins evolve slowly and play a central role within a network, a new algorithm, named CED, is proposed. CED mainly employs gene expression level, protein complex information and edge clustering coefficient to predict essential proteins. The performance of CED is validated based on the yeast Protein-Protein Interaction (PPI) network obtained from DIP database and BioGRID database. The prediction accuracy of CED outperforms other seven algorithms when applied to the two databases. PMID:26510286

  10. GTP synthases. Proton pumping and phosphorylation in ligand-receptor-G alpha-protein complexes.

    Science.gov (United States)

    Nederkoorn, P H; Timmerman, H; Donné-Op Den Kelder, G M; Timms, D; Wilkinson, A J; Kelly, D R; Broadley, K J; Davies, R H

    1996-01-01

    A structural model for a ligand-receptor-Gs alpha-protein complex to function as a GTP synthase is presented. The mechanism which is dependent on the movement and rotation of the G alpha-protein alpha 2-helix is seen to involve the delivery of, at least, one proton to the phosphorylation site in the rotation of this helix. The cycle is driven by a ligand-mediated proton pump through the alpha-helices of the receptor, attachment of the conserved Tyr-Arg-Tyr receptor proton shuttle being made to an aspartate group on the Gs alpha-protein terminal sidechain, which is itself linked to the Asn-Gln interaction known to control movement and rotation of the alpha 2-helix between .GDP and .GTP structures. The energetics of proton transfer through the shuttle mechanism and delivery of a proton to the aspartate group are shown to be sufficient to rupture this controlling interaction and its associated backbone bond. The complex leads to full spatial and energetic definition of the receptor proton shuttle mechanism, while there is a striking association of further Tyrosine and Arginine residues in the vicinity of the Gs alpha-protein Asn-Gln interaction. Calculations at the HF 6-31G** level confirm that a critical balance between ion pair and neutral forms of Tyr-Arg interactions under multiply hydrogen bonded conditions in a hydrophobic environment controls proton transfer and recovery mechanisms. The intrinsic preference of the neutral Tyr-Arg form over the ion-pair is 14.0 kcal/mol. Activation of the Tyrosine oxygen atom in the neutral form by single-NH or -OH groups reduces this difference by some 6.4-8.6 kcal/mol but the dominance of the neutral form is maintained. The expected slight overestimates are consistent with the maximum activation enthalpy of 11.0-12.0 kcal/ mol required to initiate proton transfer through the shuttle. The extended form of the shuttle with the Arginine acting competitively between the two Tyrosine residues allows interpretation of observed

  11. MDcons: Intermolecular contact maps as a tool to analyze the interface of protein complexes from molecular dynamics trajectories

    KAUST Repository

    Abdel-Azeim, Safwat

    2014-05-06

    Background: Molecular Dynamics ( MD) simulations of protein complexes suffer from the lack of specific tools in the analysis step. Analyses of MD trajectories of protein complexes indeed generally rely on classical measures, such as the RMSD, RMSF and gyration radius, conceived and developed for single macromolecules. As a matter of fact, instead, researchers engaged in simulating the dynamics of a protein complex are mainly interested in characterizing the conservation/variation of its biological interface. Results: On these bases, herein we propose a novel approach to the analysis of MD trajectories or other conformational ensembles of protein complexes, MDcons, which uses the conservation of inter-residue contacts at the interface as a measure of the similarity between different snapshots. A "consensus contact map" is also provided, where the conservation of the different contacts is drawn in a grey scale. Finally, the interface area of the complex is monitored during the simulations. To show its utility, we used this novel approach to study two protein-protein complexes with interfaces of comparable size and both dominated by hydrophilic interactions, but having binding affinities at the extremes of the experimental range. MDcons is demonstrated to be extremely useful to analyse the MD trajectories of the investigated complexes, adding important insight into the dynamic behavior of their biological interface. Conclusions: MDcons specifically allows the user to highlight and characterize the dynamics of the interface in protein complexes and can thus be used as a complementary tool for the analysis of MD simulations of both experimental and predicted structures of protein complexes.

  12. Developments in mass spectrometry for the analysis of complex protein mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Khalsa-Moyers, Gurusahai K [ORNL; McDonald, W Hayes [ORNL

    2006-01-01

    State-of-the-art proteomics workflows involve multiple interdependent steps: sample preparation, protein peptide separation, mass spectrometry and data analysis.While improvements in any of these steps can increase the depth and breadth of analysis, advances in mass spectrometry have catalysed many of the most important developments. We discuss common classes of mass analysers and how these analysers are put together to produce some of the most popular mass spectrometry platforms.The capabilities of these platforms determine how they can be used in a variety of common proteomic strategies and, in turn, what types of biological questions can be addressed. Moving forward, powerful new hybridmass spectrometers and application of emerging types of tandemmass spectrometry promise that our ability to analyse complex mixtures of proteins will continue to advance.

  13. Strengthening of the DNA-protein complex during stationary phase aging of cell cultures

    International Nuclear Information System (INIS)

    The possibility of accumulation of cross-linkages in the DNA-protein complex was studied during stationary phase aging of cells in culture. Chinese hamster cells were used in the experiments, along with human fibroblasts. 3H-thymidine, 14C-valine, and 14C-leucine were added to the medium. The quantity of protein firmly bound with DNA was judged from the value of the coefficient 14C/3H determined with allowance for penetration of counting from the 14C-channel into the 3H-channel. The authors maintain that the results presented in this paper provide further evidence of the value of stationary phase cell cultures for the study of the mechanisms of aging and also of some of the general principles underlying hereditary pathology

  14. Robustness, efficiency, and optimality in the Fenna-Matthews-Olson photosynthetic pigment-protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Lewis A.; Habershon, Scott, E-mail: S.Habershon@warwick.ac.uk [Department of Chemistry and Centre for Scientific Computing, University of Warwick, Coventry CV4 7AL (United Kingdom)

    2015-09-14

    Pigment-protein complexes (PPCs) play a central role in facilitating excitation energy transfer (EET) from light-harvesting antenna complexes to reaction centres in photosynthetic systems; understanding molecular organisation in these biological networks is key to developing better artificial light-harvesting systems. In this article, we combine quantum-mechanical simulations and a network-based picture of transport to investigate how chromophore organization and protein environment in PPCs impacts on EET efficiency and robustness. In a prototypical PPC model, the Fenna-Matthews-Olson (FMO) complex, we consider the impact on EET efficiency of both disrupting the chromophore network and changing the influence of (local and global) environmental dephasing. Surprisingly, we find a large degree of resilience to changes in both chromophore network and protein environmental dephasing, the extent of which is greater than previously observed; for example, FMO maintains EET when 50% of the constituent chromophores are removed, or when environmental dephasing fluctuations vary over two orders-of-magnitude relative to the in vivo system. We also highlight the fact that the influence of local dephasing can be strongly dependent on the characteristics of the EET network and the initial excitation; for example, initial excitations resulting in rapid coherent decay are generally insensitive to the environment, whereas the incoherent population decay observed following excitation at weakly coupled chromophores demonstrates a more pronounced dependence on dephasing rate as a result of the greater possibility of local exciton trapping. Finally, we show that the FMO electronic Hamiltonian is not particularly optimised for EET; instead, it is just one of many possible chromophore organisations which demonstrate a good level of EET transport efficiency following excitation at different chromophores. Overall, these robustness and efficiency characteristics are attributed to the highly

  15. Robustness, efficiency, and optimality in the Fenna-Matthews-Olson photosynthetic pigment-protein complex

    Science.gov (United States)

    Baker, Lewis A.; Habershon, Scott

    2015-09-01

    Pigment-protein complexes (PPCs) play a central role in facilitating excitation energy transfer (EET) from light-harvesting antenna complexes to reaction centres in photosynthetic systems; understanding molecular organisation in these biological networks is key to developing better artificial light-harvesting systems. In this article, we combine quantum-mechanical simulations and a network-based picture of transport to investigate how chromophore organization and protein environment in PPCs impacts on EET efficiency and robustness. In a prototypical PPC model, the Fenna-Matthews-Olson (FMO) complex, we consider the impact on EET efficiency of both disrupting the chromophore network and changing the influence of (local and global) environmental dephasing. Surprisingly, we find a large degree of resilience to changes in both chromophore network and protein environmental dephasing, the extent of which is greater than previously observed; for example, FMO maintains EET when 50% of the constituent chromophores are removed, or when environmental dephasing fluctuations vary over two orders-of-magnitude relative to the in vivo system. We also highlight the fact that the influence of local dephasing can be strongly dependent on the characteristics of the EET network and the initial excitation; for example, initial excitations resulting in rapid coherent decay are generally insensitive to the environment, whereas the incoherent population decay observed following excitation at weakly coupled chromophores demonstrates a more pronounced dependence on dephasing rate as a result of the greater possibility of local exciton trapping. Finally, we show that the FMO electronic Hamiltonian is not particularly optimised for EET; instead, it is just one of many possible chromophore organisations which demonstrate a good level of EET transport efficiency following excitation at different chromophores. Overall, these robustness and efficiency characteristics are attributed to the highly

  16. Robustness, efficiency, and optimality in the Fenna-Matthews-Olson photosynthetic pigment-protein complex

    International Nuclear Information System (INIS)

    Pigment-protein complexes (PPCs) play a central role in facilitating excitation energy transfer (EET) from light-harvesting antenna complexes to reaction centres in photosynthetic systems; understanding molecular organisation in these biological networks is key to developing better artificial light-harvesting systems. In this article, we combine quantum-mechanical simulations and a network-based picture of transport to investigate how chromophore organization and protein environment in PPCs impacts on EET efficiency and robustness. In a prototypical PPC model, the Fenna-Matthews-Olson (FMO) complex, we consider the impact on EET efficiency of both disrupting the chromophore network and changing the influence of (local and global) environmental dephasing. Surprisingly, we find a large degree of resilience to changes in both chromophore network and protein environmental dephasing, the extent of which is greater than previously observed; for example, FMO maintains EET when 50% of the constituent chromophores are removed, or when environmental dephasing fluctuations vary over two orders-of-magnitude relative to the in vivo system. We also highlight the fact that the influence of local dephasing can be strongly dependent on the characteristics of the EET network and the initial excitation; for example, initial excitations resulting in rapid coherent decay are generally insensitive to the environment, whereas the incoherent population decay observed following excitation at weakly coupled chromophores demonstrates a more pronounced dependence on dephasing rate as a result of the greater possibility of local exciton trapping. Finally, we show that the FMO electronic Hamiltonian is not particularly optimised for EET; instead, it is just one of many possible chromophore organisations which demonstrate a good level of EET transport efficiency following excitation at different chromophores. Overall, these robustness and efficiency characteristics are attributed to the highly

  17. Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.

    Science.gov (United States)

    Melani, Rafael D; Skinner, Owen S; Fornelli, Luca; Domont, Gilberto B; Compton, Philip D; Kelleher, Neil L

    2016-07-01

    Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric l-amino acid oxidase (∼130 kDa) and the multichain toxin cobra venom factor (∼147 kDa). The l-amino acid oxidase complex exhibited two clusters of multiproteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new antivenoms or other biotherapeutics. PMID:27178327

  18. Accurate design of megadalton-scale two-component icosahedral protein complexes.

    Science.gov (United States)

    Bale, Jacob B; Gonen, Shane; Liu, Yuxi; Sheffler, William; Ellis, Daniel; Thomas, Chantz; Cascio, Duilio; Yeates, Todd O; Gonen, Tamir; King, Neil P; Baker, David

    2016-07-22

    Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines. PMID:27463675

  19. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K [Brussels; (Trinity); (Michigan); (Stanford-MED); (Michigan-Med); (UW)

    2011-12-07

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor (β2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  20. A Complex Prime Numerical Representation of Amino Acids for Protein Function Comparison.

    Science.gov (United States)

    Chen, Duo; Wang, Jiasong; Yan, Ming; Bao, Forrest Sheng

    2016-08-01

    Computationally assessing the functional similarity between proteins is an important task of bioinformatics research. It can help molecular biologists transfer knowledge on certain proteins to others and hence reduce the amount of tedious and costly benchwork. Representation of amino acids, the building blocks of proteins, plays an important role in achieving this goal. Compared with symbolic representation, representing amino acids numerically can expand our ability to analyze proteins, including comparing the functional similarity of them. Among the state-of-the-art methods, electro-ion interaction pseudopotential (EIIP) is widely adopted for the numerical representation of amino acids. However, it could suffer from degeneracy that two different amino acid sequences have the same numerical representation, due to the design of EIIP. In light of this challenge, we propose a complex prime numerical representation (CPNR) of amino acids, inspired by the similarity between a pattern among prime numbers and the number of codons of amino acids. To empirically assess the effectiveness of the proposed method, we compare CPNR against EIIP. Experimental results demonstrate that the proposed method CPNR always achieves better performance than EIIP. We also develop a framework to combine the advantages of CPNR and EIIP, which enables us to improve the performance and study the unique characteristics of different representations. PMID:27249328

  1. The role of palmitoylation for protein recruitment to the inner membrane complex of the malaria parasite.

    Science.gov (United States)

    Wetzel, Johanna; Herrmann, Susann; Swapna, Lakshmipuram Seshadri; Prusty, Dhaneswar; John Peter, Arun T; Kono, Maya; Saini, Sidharth; Nellimarla, Srinivas; Wong, Tatianna Wai Ying; Wilcke, Louisa; Ramsay, Olivia; Cabrera, Ana; Biller, Laura; Heincke, Dorothee; Mossman, Karen; Spielmann, Tobias; Ungermann, Christian; Parkinson, John; Gilberger, Tim W

    2015-01-16

    To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.

  2. Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

    Energy Technology Data Exchange (ETDEWEB)

    Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander; Wei, Anzhi; Emanuel, Stuart L.; Gupta, Ruchira Das; Janjua, Ahsen; Cheng, Lin; Murdock, Melissa; Abramczyk, Bozena; Cohen, Daniel; Lin, Zheng; Morin, Paul; Davis, Jonathan H.; Dabritz, Michael; McLaughlin, Douglas C.; Russo, Katie A.; Chao, Ginger; Wright, Martin C.; Jenny, Victoria A.; Engle, Linda J.; Furfine, Eric; Sheriff, Steven (BMS)

    2014-10-02

    Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.

  3. Regulation of amyloid precursor protein processing by the Beclin 1 complex.

    Directory of Open Access Journals (Sweden)

    Philipp A Jaeger

    Full Text Available Autophagy is an intracellular degradation pathway that functions in protein and organelle turnover in response to starvation and cellular stress. Autophagy is initiated by the formation of a complex containing Beclin 1 (BECN1 and its binding partner Phosphoinositide-3-kinase, class 3 (PIK3C3. Recently, BECN1 deficiency was shown to enhance the pathology of a mouse model of Alzheimer Disease (AD. However, the mechanism by which BECN1 or autophagy mediate these effects are unknown. Here, we report that the levels of Amyloid precursor protein (APP and its metabolites can be reduced through autophagy activation, indicating that they are a substrate for autophagy. Furthermore, we find that knockdown of Becn1 in cell culture increases the levels of APP and its metabolites. Accumulation of APP and APP C-terminal fragments (APP-CTF are accompanied by impaired autophagosomal clearance. Pharmacological inhibition of autophagosomal-lysosomal degradation causes a comparable accumulation of APP and APP-metabolites in autophagosomes. Becn1 reduction in cell culture leads to lower levels of its binding partner Pik3c3 and increased presence of Microtubule-associated protein 1, light chain 3 (LC3. Overexpression of Becn1, on the other hand, reduces cellular APP levels. In line with these observations, we detected less BECN1 and PIK3C3 but more LC3 protein in brains of AD patients. We conclude that BECN1 regulates APP processing and turnover. BECN1 is involved in autophagy initiation and autophagosome clearance. Accordingly, BECN1 deficiency disrupts cellular autophagy and autophagosomal-lysosomal degradation and alters APP metabolism. Together, our findings suggest that autophagy and the BECN1-PIK3C3 complex regulate APP processing and play an important role in AD pathology.

  4. Architecture of Protein and DNA Contacts within the TFIIIB-DNA Complex

    OpenAIRE

    Colbert, Trenton; Lee, Sally; Schimmack, Greg; Hahn, Steven

    1998-01-01

    The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as...

  5. Grafted ionomer complexes and their effect on protein adsorption on silica and polysulfone surfaces

    OpenAIRE

    Brzozowska, A.M.; Keizer, de, O.; Detrembleur, C.; Stuart, M.A.C.; Norde, W

    2010-01-01

    We have studied the formation and the stability of ionomer complexes from grafted copolymers (GICs) in solution and the influence of GIC coatings on the adsorption of the proteins beta-lactoglobulin (beta-lac), bovine serum albumin (BSA), and lysozyme (Lsz) on silica and polysulfone. The GICs consist of the grafted copolymer PAA(28)-co-PAPEO(22) {poly(acrylic acid)-co-poly[acrylate methoxy poly(ethylene oxide)]} with negatively charged AA and neutral APEO groups, and the positively charged ho...

  6. Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene

    OpenAIRE

    Brzozowska, A.M.; Keizer, de, O.; Norde, W; Detrembleur, C.; Cohen Stuart, M. A.

    2010-01-01

    We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins beta-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer PAA(21)-b-PAPEO(14) (poly(acrylic acid)-b-poly(acrylate methoxy poly(ethylene oxide)), with a negatively charged PAA block and a neutral PAPEO block and a positively charged homopolymer P2MVPI (po...

  7. 'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex.

    OpenAIRE

    Nargang, F E; Künkele, K P; Mayer, A.; Ritzel, R G; Neupert, W; Lill, R

    1995-01-01

    MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through t...

  8. Protein kinase A governs oxidative phosphorylation kinetics and oxidant emitting potential at complex I

    Directory of Open Access Journals (Sweden)

    Daniel Stephen Lark

    2015-11-01

    Full Text Available The mitochondrial electron transport system (ETS is responsible for setting and maintaining both the energy and redox charges throughout the cell. Reversible phosphorylation of mitochondrial proteins, particularly via the soluble adenylyl cyclase (sAC/cyclic AMP (cAMP/Protein kinase A (PKA axis, has recently been revealed as a potential mechanism regulating the ETS. However, the governance of cAMP/PKA signaling and its implications on ETS function are incompletely understood. In contrast to prior reports using exogenous bicarbonate, we provide evidence that endogenous CO2 produced by increased tricarboxylic acid (TCA cycle flux is insufficient to increase mitochondrial cAMP levels, and that exogenous addition of membrane permeant 8Br-cAMP does not enhance mitochondrial respiratory capacity. We also report important non-specific effects of commonly used inhibitors of sAC which preclude their use in studies of mitochondrial function. In isolated liver mitochondria, inhibition of PKA reduces complex I-, but not complex II-supported respiratory capacity. In permeabilized myofibers, inhibition of PKA lowers both the Km and Vmax for complex I-supported respiration as well as succinate-supported H2O2 emitting potential. In summary, the data provided here improve our understanding of how mitochondrial cAMP production is regulated, illustrate a need for better tools to examine the impact of sAC activity on mitochondrial biology, and suggest that cAMP/PKA signaling contributes to the governance of electron flow through complex I of the ETS.

  9. Multipartite entanglement in the Fenna-Matthews-Olson (FMO) pigment-protein complex.

    Science.gov (United States)

    Thilagam, A

    2012-05-01

    We investigate multipartite states in the Fenna-Matthews-Olson (FMO) pigment-protein complex of the green sulfur bacteria using a Lorentzian spectral density of the phonon reservoir fitted with typical parameter estimates of the species, Prosthecochloris aestuarii. The evolution of the entanglement measure of the excitonic W qubit states is evaluated in the picosecond time range, showing increased revivals in the non-Markovian regime. Similar trends are observed in the evolution dynamics of the Meyer-Wallach measure of the N-exciton multipartite state, with results showing that multipartite entanglement can last from 0.5 to 1 ps, between the bacteriochlorophylls of the FMO complex. The teleportation and quantum information splitting fidelities associated with the Greenberger-Horne-Zeilinger and W-like resource states formed by the excitonic qubit channels of the FMO complex show that revivals in fidelities increase with the degree of non-Markovian strength of the decoherent environment. Quantum information processing tasks involving teleportation followed by the decodification process involving W-like states of the FMO complex may play a critical role during coherent oscillations at physiological temperatures. PMID:22583269

  10. biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes.

    Science.gov (United States)

    Weissmann, Florian; Petzold, Georg; VanderLinden, Ryan; Huis In 't Veld, Pim J; Brown, Nicholas G; Lampert, Fabienne; Westermann, Stefan; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

    2016-05-10

    Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes. PMID:27114506

  11. Structure and expression of major histocompatibility complex-binding protein 2, a 275-kDa zinc finger protein that binds to an enhancer of major histocompatibility complex class I genes

    NARCIS (Netherlands)

    Veer, L.J. van 't; Lutz, P.M.; Isselbacher, K.J.; Bernards, R.A.

    1992-01-01

    We have isolated a cDNA encoding a transcription factor that binds to the enhancer of major histocompatibility complex (MHC) class I genes. MHC-binding protein 2 (MBP-2) is a 275-kDa protein, containing two sets of widely separated zinc fingers and a stretch of highly acidic amino acids, a putative

  12. Modeling of kinetics of the inducible protein complexes of the SOS system in bacteria E. coli which realize TLS process

    International Nuclear Information System (INIS)

    The mathematical model describing kinetics of the inducible genes of the protein complexes, formed during SOS response in bacteria Escherichia coli is developed. Within the bounds of developed approaches the auxiliary mathematical model describing changes in concentrations of the dimers, which are the components of final protein complexes, is developed. The solutions of both models are based on the experimental data concerning expression of the basic genes of the SOS system in bacteria Escherichia coli

  13. PRISM: a web server and repository for prediction of protein–protein interactions and modeling their 3D complexes

    OpenAIRE

    Baspinar, Alper; Cukuroglu, Engin; Nussinov, Ruth; Keskin, Ozlem; Gursoy, Attila

    2014-01-01

    The PRISM web server enables fast and accurate prediction of protein–protein interactions (PPIs). The prediction algorithm is knowledge-based. It combines structural similarity and accounts for evolutionary conservation in the template interfaces. The predicted models are stored in its repository. Given two protein structures, PRISM will provide a structural model of their complex if a matching template interface is available. Users can download the complex structure, retrieve the interface r...

  14. SLIDE, the Protein Interacting Domain of Imitation Switch Remodelers, Binds DDT-Domain Proteins of Different Subfamilies in Chromatin Remodeling Complexes

    Institute of Scientific and Technical Information of China (English)

    Jiaqiang Dong; Zheng Gao; Shujing Liu; Guang Li; Zhongnan Yang; Hai Huang; Lin Xu

    2013-01-01

    The Imitation Switch (ISWI) type adenosine triphosphate (ATP)-dependent chromatin remodeling factors are conserved proteins in eukaryotes, and some of them are known to form stable remodeling complexes with members from a family of proteins, termed DDT-domain proteins. Although it is well documented that ISWIs play important roles in different biological processes in many eukaryotic species, the molecular basis for protein interactions in ISWI complexes has not been fully addressed. Here, we report the identification of interaction domains for both ISWI and DDT-domain proteins. By analyzing CHROMATIN REMODELING11 (CHR11) and RINGLET1 (RLT1), an Arabidopsis thaliana ISWI (AtISWI) and AtDDT-domain protein, respectively, we show that the SLIDE domain of CHR11 and the DDT domain together with an adjacent sequence of RLT1 are responsible for their binding. The Arabidopsis genome contains at least 12 genes that encode DDT-domain proteins, which could be grouped into five subfamilies based on the sequence similarity. The SLIDE domain of AtISWI is able to bind members from different AtDDT subfamilies. Moreover, a human ISWI protein SNF2H is capable of binding AtDDT-domain proteins through its SLIDE domain, suggesting that binding to DDT-domain proteins is a conserved biochemical function for the SLIDE domain of ISWIs in eukaryotes.

  15. A sulfhydryl-reactive ruthenium (II complex and its conjugation to protein G as a universal reagent for fluorescent immunoassays.

    Directory of Open Access Journals (Sweden)

    Jing-Tang Lin

    Full Text Available To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2'-dipyridine Ruthenium bis (hexafluorophosphate. The synthesized Ru(II complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. The emission peak wavelength of the Ru(II-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II complex, indicating that Ru(II-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG binding assay was conducted. The result showed that Ru(II-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.

  16. Structure of a C-terminal fragment of its Vps53 subunit suggests similarity of Golgi-associated retrograde protein (GARP) complex to a family of tethering complexes

    Energy Technology Data Exchange (ETDEWEB)

    Vasan, Neil; Hutagalung, Alex; Novick, Peter; Reinisch, Karin M. (Yale); (UCLJ)

    2010-08-13

    The Golgi-associated retrograde protein (GARP) complex is a membrane-tethering complex that functions in traffic from endosomes to the trans-Golgi network. Here we present the structure of a C-terminal fragment of the Vps53 subunit, important for binding endosome-derived vesicles, at a resolution of 2.9 {angstrom}. We show that the C terminus consists of two {alpha}-helical bundles arranged in tandem, and we identify a highly conserved surface patch, which may play a role in vesicle recognition. Mutations of the surface result in defects in membrane traffic. The fold of the Vps53 C terminus is strongly reminiscent of proteins that belong to three other tethering complexes - Dsl1, conserved oligomeric Golgi, and the exocyst - thought to share a common evolutionary origin. Thus, the structure of the Vps53 C terminus suggests that GARP belongs to this family of complexes.

  17. The Rieske Fe/S protein of the cytochrome b6/f complex in chloroplasts: missing link in the evolution of protein transport pathways in chloroplasts?

    Science.gov (United States)

    Molik, S; Karnauchov, I; Weidlich, C; Herrmann, R G; Klösgen, R B

    2001-11-16

    The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b(6)/f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the DeltapH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH(2)-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of DeltapH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways. PMID:11526115

  18. Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.

    Science.gov (United States)

    Boratkó, Anita; Péter, Margit; Thalwieser, Zsófia; Kovács, Előd; Csortos, Csilla

    2015-12-01

    TIMAP (TGF-β inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements. PMID:26497934

  19. Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA-protein complexes.

    Science.gov (United States)

    McAndrew, Christopher P; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L; Pegg, Ian L; Sarkar, Abhijit

    2016-01-01

    We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA-protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether's extension: ~0.05 pN in force and DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule's force response. Using a calibration technique based on Stokes' drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level.

  20. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.

    2010-05-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  1. Self-assembly and Evolution from protein complexes to DNA nanostructures

    Science.gov (United States)

    Louis, Ard A.

    2012-02-01

    The remarkable ability of biological matter to robustly self-assemble into well defined composite objects excites the imagination, suggesting that these processes could perhaps be emulated through the judicious design of synthetic building blocks. We use statistical mechanics to uncover the design rules for self-assembly into well defined three dimensional composite objects. In Nature, the rules for self-assembly emerge from an evolutionary process. We show how some patterns in protein complexes can be explained by their evolutionary origin [1]. We also introduce a coarse-grained rigid nucleotide model of DNA that reproduces the basic thermodynamics of short strands: duplex hybridization, single-stranded stacking and hairpin formation, and also captures the essential structural properties of DNA: the helical pitch, persistence length and torsional stiffness of double-stranded molecules, as well as the comparative flexibility of unstacked single strands [2]. We apply the model to calculate the detailed free-energy landscape of one full cycle of DNA ``tweezers,'' a simple machine driven by hybridization and strand displacement. We also study other nanomachines as well as processes such as force-induced melting, cruciform formation and the self-assembly of DNA tetrahedra.[4pt] [1] The self-assembly and evolution of homomeric protein complexes Gabriel Villar, et al., Phys. Rev. Lett. 102, 118106 (2009[0pt] [2] Structural and thermodynamic properties of a coarse-grained model of DNA, Thomas E. Ouldridge, Ard A. Louis, Jonathan P.K. Doye, J. Chem. Phys. 134 , 085101 (2011)

  2. Photosynthate Regulation of the Root System Architecture Mediated by the Heterotrimeric G Protein Complex in Arabidopsis.

    Science.gov (United States)

    Mudgil, Yashwanti; Karve, Abhijit; Teixeira, Paulo J P L; Jiang, Kun; Tunc-Ozdemir, Meral; Jones, Alan M

    2016-01-01

    Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gβ subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior. PMID:27610112

  3. Rodlike Complexes of a Polyelectrolyte (Hyaluronan) and a Protein (Lysozyme) observed by SANS

    CERN Document Server

    Boué, François; Cousin, Fabrice; Grillo, Isabelle; Morfin, Isabelle; 10.1021/bm100861g

    2012-01-01

    We study by Small Angle Neutron Scattering (SANS) the structure of Hyaluronan -Lysozyme complexes. Hyaluronan (HA) is a polysaccharide of 9 nm intrinsic persistence length that bears one negative charge per disaccharide monomer (Mmol = 401.3 g/mol); two molecular weights, Mw = 6000 and 500 000 Da were used. The pH was adjusted at 4.7 and 7.4 so that lysozyme has a global charge of +10 and + 8 respectively. The lysozyme concentration was varied from 3 to 40 g/L, at constant HA concentration (10 g/L). At low protein concentration, samples are monophasic and SANS experiments reveal only fluctuations of concentration although, at high protein concentration, clusters are observed by SANS in the dense phase of the diphasic samples. In between, close to the onset of the phase separation, a distinct original scattering is observed. It is characteristic of a rod-like shape, which could characterize "single" complexes involving one or a few polymer chains. For the large molecular weight (500 000) the rodlike rigid doma...

  4. Prediction of TF target sites based on atomistic models of protein-DNA complexes

    Directory of Open Access Journals (Sweden)

    Collado-Vides Julio

    2008-10-01

    Full Text Available Abstract Background The specific recognition of genomic cis-regulatory elements by transcription factors (TFs plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence. Results Here we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models. Conclusion Our results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition.

  5. Crystallization of Mitochondrial Respiratory Complex II fromChicken Heart: A Membrane-Protein Complex Diffracting to 2.0Angstrom

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward A.

    2004-12-17

    Procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Angstrom with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.

  6. DMPD: Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complexes: a short review. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 1373512 Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complex....html) (.csml) Show Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complex...ride (LPS)-binding protein (LBP) and CD14, thereceptor for LPS/LBP complexes: a short review. Authors Schuma

  7. Structures of Neuroligin-1 And the Neuroligin-1/Neurexin-1beta Complex Reveal Specific Protein-Protein And Protein-Ca**2+ Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Arac, D.; Boucard, A.A.; Ozkan, E.A; Strop, P.; Newell, E.; Sudhof, T.C.; Brunger, A.T.

    2009-05-28

    Neurexins and neuroligins provide trans-synaptic connectivity by the Ca{sup 2+}-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1{beta}. Neuroligin-1 forms a constitutive dimer, and two neurexin-1{beta} monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1{beta} complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca{sup 2+}. Alternatively spliced sites in neurexin-1{beta} and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1{beta}, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1{beta} complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

  8. Structures of Neuroligin-1 And the Neuroligin-1/Neurexin-1 Beta Complex Reveal Specific Protein-Protein And Protein-Ca2+ Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Demet, Arac; Boucard, A.A.; Ozkan, E.A; Strop, P.; Newell, E.; Sudhof, T.C.; Brunger, A.T.

    2009-06-01

    Neurexins and neuroligins provide trans-synaptic connectivity by the Ca{sup 2+}-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1{beta}. Neuroligin-1 forms a constitutive dimer, and two neurexin-1{beta} monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1{beta} complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca{sup 2+}. Alternatively spliced sites in neurexin-1{beta} and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1{beta}, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1{beta} complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

  9. Long-lifetime Ru(II) complexes for the measurement of high molecular weight protein hydrodynamics.

    Science.gov (United States)

    Szmacinski, H; Castellano, F N; Terpetschnig, E; Dattelbaum, J D; Lakowicz, J R; Meyer, G J

    1998-03-01

    We describe the synthesis and characterization of two asymmetrical ruthenium(II) complexes, [Ru(dpp)2(dcbpy)]2+ and [Ru(dpp)2(mcbpy)]2+, as well as the water soluble sulfonated derivatives [Ru(dpp(SO3Na)2)2(dcbpy)]2+ and [Ru(dpp(SO3Na)2)2(mcbpy)]2+ (dpp is 4,7-diphenyl-1,10-phenanthroline, dcbpy is 4,4'-dicarboxylic acid-2,2'-bipyridine, mcbpy is 4-methyl,4'-carboxylic acid-2,2'-bipyridine, and dpp(SO3Na)2 is the disulfonated derivative of dpp) as probes for the measurement of the rotational motions of proteins. The spectral (absorption, emission, and anisotropy) and photophysical (time-resolved intensity and anisotropy decays) properties of these metal-ligand complexes were determined in solution, in both the presence and absence of human serum albumin (HSA). These complexes display lifetimes ranging from 345 ns to 3.8 microseconds in deoxygenated aqueous solutions under a variety of conditions. The carboxylic acid groups on these complexes were activated to form N-hydroxysuccinimide (NHS) esters which were used to covalently lable HSA, and were characterized spectroscopically in the same manner as above. Time-resolved anisotropy measurements were performed to demonstrate the utility of these complexes in measuring long rotational correlation times of bioconjugates between HSA and antibody to HSA. The potential usefulness of these probes in fluorescence polarization immunoassays was demonstrated by an association assay of the Ru(II)-labeled HSA with polyclonal antibody. PMID:9546056

  10. Structure of the major membrane protein complex from urinary bladder epithelial cells by cryo-electron crystallography

    NARCIS (Netherlands)

    Oostergetel, GT; Keegstra, W; Brisson, A

    2001-01-01

    Numerous protein plaques cover the apical surface of mammalian urinary bladder epithelial cells. These plaques contain four integral membrane proteins, called uroplakins, which form a well-ordered array of hexameric complexes. The 3D structure of these naturally occurring 2D crystals was studied by

  11. Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

    Science.gov (United States)

    Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

    2016-01-01

    In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

  12. Grafted block complex coacervate core micelles and their effect on protein adsorption on silica and polystyrene.

    Science.gov (United States)

    Brzozowska, Agata M; de Keizer, Arie; Norde, Willem; Detrembleur, Christophe; Cohen Stuart, Martien A

    2010-07-01

    We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins beta-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer PAA(21)-b-PAPEO(14) (poly(acrylic acid)-b-poly(acrylate methoxy poly(ethylene oxide)), with a negatively charged PAA block and a neutral PAPEO block and a positively charged homopolymer P2MVPI (poly(N-methyl 2-vinyl pyridinium iodide). In solution, these C3Ms partly disintegrate at salt concentrations between 50 and 100 mM NaCl. Adsorption of C3Ms and proteins has been studied with fixed-angle optical reflectometry, at salt concentrations ranging from 1 to 100 mM NaCl. In comparison with the adsorption of PAA(21)-b-PAPEO(14) alone adsorption of C3Ms significantly increases the amount of PAA(21)-b-PAPEO(14) on the surface. This results in a higher surface density of PEO chains. The stability of the C3M coatings and their influence on protein adsorption are determined by the composition and the stability of the C3Ms in solution. A C3M-PAPEO(14)/P2MVPI(43) coating strongly suppresses the adsorption of all proteins on silica and polystyrene. The reduction of protein adsorption is the highest at 100 mM NaCl (>90%). The adsorbed C3M-PAPEO(14)/P2MVPI(43) layer is partly removed from the surface upon exposure to an excess of beta-lactoglobulin solution, due to formation of soluble aggregates consisting of beta-lactoglobulin and P2MVPI(43). In contrast, C3M-PAPEO(14)/P2MVPI(228) which has a fivefold longer cationic block enhances adsorption of the negatively charged proteins on both surfaces at salt concentrations above 1 mM NaCl. A single PAA(21)-b-PAPEO(14) layer causes only a moderate reduction of protein adsorption.

  13. Outer Membrane Protein Complex of Meningococcus Enhances the Antipolysaccharide Antibody Response to Pneumococcal Polysaccharide–CRM197 Conjugate Vaccine ▿

    OpenAIRE

    Lai, Zengzu; Schreiber, John R.

    2011-01-01

    Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antib...

  14. P105 and p100 proteins function as the core of heterogeneous NF-kappaB complexes

    OpenAIRE

    Savinova, Olga V.

    2009-01-01

    Biochemistry of the components of signal transduction pathways provides a foundation for the understanding of how cellular signaling events initiate, propagate, and terminate. This thesis describes the multi-protein heterogeneous complexes of NF-kappaB with p105 and p100 proteins. Chapter 1 introduces the NF-kappaB signaling pathway and provides the rational for this thesis research. Chapter 2 describes the results of biochemical characterization of endogenous NF-kappaB and IkappaB proteins t...

  15. RhoGTPase-binding proteins, the exocyst complex and polarized vesicle trafficking.

    Science.gov (United States)

    Mukherjee, Debarati; Sen, Arpita; Aguilar, R Claudio

    2014-01-01

    Cell polarity, the asymmetric distribution of proteins and lipids, is essential for a variety of cellular functions. One mechanism orchestrating cell polarity is polarized vesicle trafficking; whereby cargo loaded secretory vesicles are specifically transported to predetermined areas of the cell. The evolutionarily conserved exocyst complex and its small GTPase regulators play crucial roles in spatiotemporal control of polarized vesicle trafficking. In studies on neuronal membrane remodeling and synaptic plasticity, conserved mechanisms of exocyst regulation and cargo recycling during polarized vesicle trafficking are beginning to emerge as well. Recently, our lab demonstrated that RhoGTPase-binding proteins in both yeast (Bem3) and mammals (Ocrl1) are also required for the efficient traffic of secretory vesicles to sites of polarized growth and signaling. Together with our studies, we highlight the evolutionary conservation of the basic elements essential for polarized vesicle traffic across different cellular functions and model systems. In conclusion, we emphasize that studies on RhoGTPase-binding proteins in these processes should be included in the next level of investigation, for a more complete understanding of their hitherto unknown roles in polarized membrane traffic and exocyst regulation.

  16. Ku protein complex is involved in nucleotide excision repair of DNA

    International Nuclear Information System (INIS)

    The repair of ultraviolet light (UV-C, 254 nm) DNA lesions by nucleotide excision repair (NER) has been studied in the rodent cell line xrs6 belonging to complementation group 5 of ionising radiation sensitive (IRs) mutants. xrs6 cell line shows e defect in he DNA-end binding protein complex Ku which is involved in the repair of double-strand breaks (DSB) due to IR. In agreement with IR sensitivity, a bleomycin sensitive phenotype of xrs6 cell line was found as compared to the parental CHO-Kl line (factor> 8 fold). xrs6 exhibited also a slight (factor 2) but reproducible sensitivity to UV-C-light, while a revertant cell line for Ku DNA-end binding activity, xrs6rev, showed a restoration of both IR and UV-C sensitivities to the parental level. The NER activity of these cell lines was measured in vitro in nuclear protein extracts in the presence of plasmid DNA repair substrate damaged with UV-C lesions repaired by NER: xrs6 cell extracts exhibited only 55 % of NER activity as compared to the control CHO-Kl and xrs6rev cell extracts. These indicate that the Ku DSB repair protein in involved also in the NER process. (authors). 31 refs., 1 fig., 1 tab

  17. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    Energy Technology Data Exchange (ETDEWEB)

    Manno, D; Filippo, E; Fiore, R; Serra, A [Dipartimento di Scienza dei Materiali, Universita del Salento, Lecce (Italy); Urso, E; Rizzello, A; Maffia, M [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita del Salento, Lecce (Italy)

    2010-04-23

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP{sup C}. In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP{sup C} at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  18. The Leishmania donovani lipophosphoglycan T lymphocyte-reactive component is a tightly associated protein complex.

    Science.gov (United States)

    Jardim, A; Tolson, D L; Turco, S J; Pearson, T W; Olafson, R W

    1991-11-15

    Lymphocytes from mice immunized with Leishmania donovani (LPG) were specifically stimulated to proliferate in vitro by purified LPG or its delipidated congener, phosphoglycan. The response was dose dependent and required prior immunization with either LPG or phosphoglycan. Proliferation was eliminated by specific depletion of Thy-1+ cells with antisera and C and the proliferating T cell subset was shown to be CD4+CD8-. Tests of various LPG fragments indicated that the T cell stimulation was associated with the core structure of LPG rather than the lipid or phosphoglycan repeat structure. However, amino acid analysis of LPG and active LPG fragments, after acid hydrolysis, showed the presence of amino acids in peptide linkage. Specific hydrolysis of the glycosidic linkages in LPG with trifluoromethanesulfonic acid provided polypeptide material reactive with two mAb previously believed to be LPG carbohydrate core specific. The protein was separated from LPG by reverse phase chromatography and shown to be a complex of proteins with common epitopes recognized by the two mAb. The dominant species isolated from LPG was a set of small, approximately 11,000 Mr, molecules. Subsequent T cell proliferation studies showed that the lymphocyte stimulation was associated with the protein component of LPG and not the glycan. PMID:1940354

  19. Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Huaying, E-mail: zhaoh3@mail.nih.gov; Schuck, Peter, E-mail: zhaoh3@mail.nih.gov [National Institutes of Health, Bethesda, MD 20892 (United States)

    2015-01-01

    Global multi-method analysis for protein interactions (GMMA) can increase the precision and complexity of binding studies for the determination of the stoichiometry, affinity and cooperativity of multi-site interactions. The principles and recent developments of biophysical solution methods implemented for GMMA in the software SEDPHAT are reviewed, their complementarity in GMMA is described and a new GMMA simulation tool set in SEDPHAT is presented. Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design.

  20. Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT

    International Nuclear Information System (INIS)

    Global multi-method analysis for protein interactions (GMMA) can increase the precision and complexity of binding studies for the determination of the stoichiometry, affinity and cooperativity of multi-site interactions. The principles and recent developments of biophysical solution methods implemented for GMMA in the software SEDPHAT are reviewed, their complementarity in GMMA is described and a new GMMA simulation tool set in SEDPHAT is presented. Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design

  1. Multipartite entanglement in the Fenna-Matthews-Olson (FMO) pigment-protein complex

    CERN Document Server

    Thilagam, A

    2012-01-01

    We investigate the parameters involved in the long coherence times associated with multipartite states in the Fenna-Matthews-Olson (FMO) pigment-protein complex of the green sulfur bacteria. Using the time-convolutionless (TCL) projection operator technique and a Lorentzian spectral density of the phonon reservoir fitted with typical parameter estimates of the FMO complex species, {P. aestuarii}, the evolution of the entanglement measure of the excitonic qubit W states is evaluated in the picosecond time range, showing increased revivals of the entanglement lasting up to 0.4 ps in the non-Markovian regime. Similar trends are observed in the evolution dynamics of the Meyer-Wallach measure of the N-excitonic qubit multipartite state, with results showing that multipartite entanglement can last up to at least 0.5 ps,between the Bchls of the FMO complex in a decoherent environment and at cryogenic temperatures. Finally the teleportation and quantum information splitting fidelities [Chaves et. al., Phys. Rev. A 82...

  2. Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

    Science.gov (United States)

    Jonas, Kim C; Huhtaniemi, Ilpo; Hanyaloglu, Aylin C

    2016-01-01

    The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling.

  3. Transmembrane electric potential difference in the protein-pigment complex of photosystem 2.

    Science.gov (United States)

    Mamedov, M D; Kurashov, V N; Petrova, I O; Semenov, A Yu

    2012-09-01

    The protein-pigment complex of photosystem 2 (PS2) localized in the thylakoid membranes of higher plants, algae, and cyanobacteria is the main source of oxygen on Earth. The light-induced functioning of PS2 is directly linked to electron and proton transfer across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). The major contribution to ΔΨ of the PS2 reaction center is due to charge separation between the primary chlorophyll donor P(680) and the quinone acceptor Q(A), accompanied by re-reduction of P(680)(+) by the redox-active tyrosine residue Y(Z). The processes associated with the uptake and release of protons on the acceptor and donor sides of the enzyme, respectively, are also coupled with ΔΨ generation. The objective of this work was to describe the mechanisms of ΔΨ generation associated with the S-state transitions of the water-oxidizing complex in intact PS2 complex and in PS2 preparation depleted of Mn(4)Ca cluster in the presence of artificial electron donors. The findings elucidate the mechanisms of electrogenic reactions on the PS2 donor side and may be a basis for development of an effective solar energy conversion system. PMID:23157254

  4. Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein

    International Nuclear Information System (INIS)

    The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous α and β subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length λCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal α-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the β-β interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the α-RNAP of the pTRG vector and λCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the β-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the β-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the β-subunits of the native MoFe protein in Bacterio

  5. On docking, scoring and assessing protein-DNA complexes in a rigid-body framework.

    Directory of Open Access Journals (Sweden)

    Marc Parisien

    Full Text Available We consider the identification of interacting protein-nucleic acid partners using the rigid body docking method FTdock, which is systematic and exhaustive in the exploration of docking conformations. The accuracy of rigid body docking methods is tested using known protein-DNA complexes for which the docked and undocked structures are both available. Additional tests with large decoy sets probe the efficacy of two published statistically derived scoring functions that contain a huge number of parameters. In contrast, we demonstrate that state-of-the-art machine learning techniques can enormously reduce the number of parameters required, thereby identifying the relevant docking features using a miniscule fraction of the number of parameters in the prior works. The present machine learning study considers a 300 dimensional vector (dependent on only 15 parameters, termed the Chemical Context Profile (CCP, where each dimension reflects a specific type of protein amino acid-nucleic acid base interaction. The CCP is designed to capture the chemical complementarities of the interface and is well suited for machine learning techniques. Our objective function is the Chemical Context Discrepancy (CCD, which is defined as the angle between the native system's CCP vector and the decoy's vector and which serves as a substitute for the more commonly used root mean squared deviation (RMSD. We demonstrate that the CCP provides a useful scoring function when certain dimensions are properly weighted. Finally, we explore how the amino acids on a protein's surface can help guide DNA binding, first through long-range interactions, followed by direct contacts, according to specific preferences for either the major or minor grooves of the DNA.

  6. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1–AMPK complex

    International Nuclear Information System (INIS)

    Highlights: ► The nuclear protein Artemis physically interacts with AMPKα2. ► Artemis co-localizes with AMPKα2 in the nucleus. ► Artemis promotes phosphorylation and activation of AMPK. ► The interaction between AMPKα2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory β and γ subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the α-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPKα2-binding protein. Artemis was found to co-immunoprecipitate with AMPKα2, and the co-localization of Artemis with AMPKα2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPKα2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPKα2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1–AMPK complex.

  7. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex

    Energy Technology Data Exchange (ETDEWEB)

    Nakagawa, Koji, E-mail: k_nakagawa@pharm.hokudai.ac.jp [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Asaka, Masahiro [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Takeda, Hiroshi [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Kobayashi, Masanobu [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); School of Nursing and Social Services, Health Sciences University of Hokkaido, Ishikari-Toubetsu, Hokkaido 061-0293 (Japan)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

  8. Protein phosphatases and chromatin modifying complexes in the inflammatory cascade in acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Javier; Escobar; Javier; Pereda; Alessandro; Arduini; Juan; Sastre; Juan; Sandoval; Luis; Aparisi; Gerardo; López-Rodas; Luis; Sabater

    2010-01-01

    Acute pancreatitis is an inflammation of the pancreas that may lead to systemic inflammatory response syndrome and death due to multiple organ failure. Acinar cells, together with leukocytes, trigger the inflammatory cascade in response to local damage of the pancreas. Amplification of the inflammatory cascade requires up-regulation of proinflammatory cytokines and this process is mediated not only by nuclear factor κB but also by chromatinmodifying complexes and chromatin remodeling. Among the different families of histone acetyltransferases, the p300/CBP family seems to be particularly associated with the inflammatory process. cAMP activates gene expression via the cAMP-responsive element (CRE) and the transcription factor CRE-binding protein (CREB). CREB can be phosphorylated and activated by different kinases, such as protein kinase A and MAPK, and then it recruits the histone acetyltransferase co-activator CREB-binding protein (CBP) and its homologue p300. The recruitment of CBP/p300 and changes in the level of histone acetylation are required for transcription activation. Transcriptional repression is also a dynamic and essential mechanism of down-regulation of genes for resolution of inflammation, which seems to be mediated mainly by protein phosphatases (PP1, PP2A and MKP1) and histone deacetylases(HDACs) .Class HDACs are key transcriptional regulators whose activities are controlled via phosphorylationdependent nucleo/cytoplasmic shuttling. PP2A is responsible for dephosphorylation of class HDACs, triggeringnuclear localization and repression of target genes, whereas phosphorylation triggers cytoplasmic localization leading to activation of target genes. The potential benefit from treatment with phosphodiesterase inhibitors and histone deacetylase inhibitors is discussed.

  9. A light-harvesting siphonaxanthin-chlorophyll a/b-protein complex of marine green alga,Bryopsis corticulans

    Institute of Scientific and Technical Information of China (English)

    CHEN Hui; SHEN Shihua; HE Junfang; LENG Jing; LI Liangbi; KUANG Tingyun

    2004-01-01

    A light-harvesting chlorophyll a/b-protein complex (LHCP) was isolated directly from thylakoid membranes of marine green alga, Bryopsis corticulans, by two consecutive runs of liquid chromatography. The trimeric form of the light-harvesting complex has been obtained by sucrose gradient ultracentrifugation. The result of SDSPAGE shows that the light-harvesting complex is composed of at least five apoproteins in which a protein with apparent molecular weight of about 31 kD was never found in the major light-harvesting complex (LHC Ⅱ) from higher plants.The isolated Bryopsis corticulans light-harvesting complex contains a specific carotenoid, siphonaxanthin, as well as chlorophyll (Chl) a, Chl b, neoxanthin and violaxanthin. Siphonaxanthin which is present in the light-harvesting siphonaxanthin-chlorophyll a/b-protein complex of Bryopsis corticulans is responsible for enhanced absorption in the blue-green region (530 nm). Efficient energy transfer from both siphonaxanthin and Chl b to Chl a in Bryopsis corticulans LHCP, which has similar absorption and fluorescence emission spectra to those of the lutein-chlorophyll a/b-protein of higher plants, proved that molecular arrangement of the light-harvesting pigments was highly ordered in the Bryopsis corticulans LHCP. The siphonaxanthin-chlorophyll a/b-proteins allow enhanced absorption of blue-green light, the predominant light available in deep ocean waters or shaded subtidal marine habitats.

  10. Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis

    Directory of Open Access Journals (Sweden)

    Bernardo Blanco-Sánchez

    2014-05-01

    Full Text Available Usher syndrome (USH, the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER. Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.

  11. A Polypyrimidine Tract Binding Protein, Pumpkin RBP50, Forms the Basis of a Phloem-Mobile Ribonucleoprotein Complex[W

    Science.gov (United States)

    Ham, Byung-Kook; Brandom, Jeri L.; Xoconostle-Cázares, Beatriz; Ringgold, Vanessa; Lough, Tony J.; Lucas, William J.

    2009-01-01

    RNA binding proteins (RBPs) are integral components of ribonucleoprotein (RNP) complexes and play a central role in RNA processing. In plants, some RBPs function in a non-cell-autonomous manner. The angiosperm phloem translocation stream contains a unique population of RBPs, but little is known regarding the nature of the proteins and mRNA species that constitute phloem-mobile RNP complexes. Here, we identified and characterized a 50-kD pumpkin (Cucurbita maxima cv Big Max) phloem RNA binding protein (RBP50) that is evolutionarily related to animal polypyrimidine tract binding proteins. In situ hybridization studies indicated a high level of RBP50 transcripts in companion cells, while immunolocalization experiments detected RBP50 in both companion cells and sieve elements. A comparison of the levels of RBP50 present in vascular bundles and phloem sap indicated that this protein is highly enriched in the phloem sap. Heterografting experiments confirmed that RBP50 is translocated from source to sink tissues. Collectively, these findings established that RBP50 functions as a non-cell-autonomous RBP. Protein overlay, coimmunoprecipitation, and cross-linking experiments identified the phloem proteins and mRNA species that constitute RBP50-based RNP complexes. Gel mobility-shift assays demonstrated that specificity, with respect to the bound mRNA, is established by the polypyrimidine tract binding motifs within such transcripts. We present a model for RBP50-based RNP complexes within the pumpkin phloem translocation stream. PMID:19122103

  12. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

    Science.gov (United States)

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-09-01

    Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

  13. Differential localization of SNARE complex proteins SNAP-25, syntaxin, and VAMP during development of the mammalian retina.

    Science.gov (United States)

    Greenlee, M H; Roosevelt, C B; Sakaguchi, D S

    2001-02-12

    SNARE complex proteins have critical functions during regulated vesicular release of neurotransmitter. In addition, they play critical roles during neurite outgrowth and synaptogenesis. Although it is clear that the function of any one SNARE complex protein during release of neurotransmitter is dependent on its association with other members of the complex, it is less certain whether their function during development and differentiation is dependent on interaction with one another. Previously, we have observed transient high levels of SNARE complex protein SNAP-25 in developing cholinergic amacrine cells (West Greenlee et al. [1998] J Comp Neurol 394:374-385). In addition, we detected, high levels of SNAP-25 in developing and mature photoreceptors. To better understand the functional significance of these high levels of SNAP-25 expression, we used immunocytochemistry to examine the developmental expression of the three members of the SNARE complex, SNAP-25, Syntaxin, and vesicle associated membrane protein (VAMP/also Synaptobrevin). Our results demonstrate that the high levels of SNAP-25 in cholinergic amacrine cells and photoreceptors are not accompanied by the same relatively high levels of other SNARE complex proteins. These results suggest that high levels of SNAP-25 in specific cell types may function independently of association with Syntaxin and VAMP. In this analysis, we characterized the changing patterns of immunoreactivity for the three SNARE complex proteins during the development and differentiation of the mammalian retina. We have compared the pattern of expression of the core SNARE complex proteins in the Brazilian opossum, Monodelphis domestica, and in the rat and found common patterns of expression between these diverse mammalian species. We observed temporal differences in the onset of immunoreactivity between these three proteins, and differences in their localization within synaptic layers in the developing and mature mammalian retina. This

  14. Complexity

    CERN Document Server

    Gershenson, Carlos

    2011-01-01

    The term complexity derives etymologically from the Latin plexus, which means interwoven. Intuitively, this implies that something complex is composed by elements that are difficult to separate. This difficulty arises from the relevant interactions that take place between components. This lack of separability is at odds with the classical scientific method - which has been used since the times of Galileo, Newton, Descartes, and Laplace - and has also influenced philosophy and engineering. In recent decades, the scientific study of complexity and complex systems has proposed a paradigm shift in science and philosophy, proposing novel methods that take into account relevant interactions.

  15. In situ quantification of HER2–protein tyrosine kinase 6 (PTK6) protein–protein complexes in paraffin sections from breast cancer tissues

    Science.gov (United States)

    Aubele, M; Spears, M; Ludyga, N; Braselmann, H; Feuchtinger, A; Taylor, K J; Lindner, K; Auer, G; Stering, K; Höfler, H; Schmitt, M; Bartlett, J M S

    2010-01-01

    Background: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. Method and results: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6–HER2 protein–protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). Conclusion: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2–PTK6 complexes are of prognostic relevance. PMID:20700126

  16. S-Adenosylmethionine conformations in solution and in protein complexes: Conformational influences of the sulfonium group

    DEFF Research Database (Denmark)

    Markham, George D.; Norrby, Per-Ola; Bock, Charles W.

    2002-01-01

    S-Adenosylmethionine (AdoMet) and other sulfonium ions play central roles in the metabolism of all organisms. The conformational preferences of AdoMet and two other biologically important sulfonium ions, S-methylmethionine and dimethylsulfonioproprionic acid, have been investigated by NMR...... and computational studies. Molecular mechanics parameters for the sulfonium center have been developed for the AMBER force field to permit analysis of NMR results and to enable comparison of the relative energies of the different conformations of AdoMet that have been found in crystal structures of complexes...... with proteins. S-Methylmethionine and S-dimethylsulfonioproprionate adopt a variety of conformations in aqueous solution; a conformation with an electrostatic interaction between the sulfonium sulfur and the carboxylate group is not noticeably favored, in contrast to the preferred conformation found by in vacuo...

  17. Modelling small-angle scattering data from complex protein-lipid systems

    DEFF Research Database (Denmark)

    Kynde, Søren Andreas Røssell

    are particularly interesting to study because they are common targets for pharmaceutical drugs. At the same time they are unfortunately unstable in solution which make them challenging to study. Phospholipid nanodiscs are small patches of lipid membrane stabilised by a belt of amphipathic helices. They can act...... as carriers of membrane proteins. Together they form monodisperse soluble aggregates of about 10 nm in size. Chapter 2 introduces the method of small-angle scattering. Small-angle X-ray and neutron scattering are well suited for studying particles in solution on length scales from 1 to 100 nm. This makes...... combine the bene ts of each of the methods and give unique structural information about relevant bio-molecular complexes in solution. Chapter 4 describes the work behind a proposal of a small-angle neutron scattering instrument for the European Spallation Source under construction in Lund. The instrument...

  18. Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rynditch A. V.

    2012-12-01

    Full Text Available Intersectin 1 (ITSN1 is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co-precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co-localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells.

  19. Origin of long-lived coherence and excitation dynamics in pigment-protein complexes

    CERN Document Server

    Zhang, Zhedong

    2015-01-01

    We uncover the mechanism of long-lived coherence that the discrete vibrational modes effectively weaken the exciton-environment interaction. This subsequently demonstrates the role of vibrational coherence which greatly contributes to long-lived feature of the excitonic coherence that has been observed in femtosecond experiments. To test the validity of our effective theory, we study the pigment-protein complex in details by exploring the energy transfer and coherence dynamics. The ground-state coherence generated by incoherent radiations is demonstrated to be significant to promote the excitation energy transfer. This on the other hand, seems to be natural from the point of view of nonequilibriumness, which funnels the downhill immigration of excitons. Moreover we also confirm that the considerable improvement of energy transfer is always accompanied by the long-lived oscillation of coherence.

  20. The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants

    Directory of Open Access Journals (Sweden)

    Madlen eNietzsche

    2014-02-01

    Full Text Available In plants, SNF1-related kinase (SnRK1 responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic alpha subunit associates with a regulatory beta subunit and an activating gamma subunit. Several different metabolites as well as the hormone abscisic acid (ABA have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF 581 could interact with both isoforms of the SnRK1 alpha subunit (AKIN10/11 of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins can share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.

  1. The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

    Energy Technology Data Exchange (ETDEWEB)

    Thompson,J.; Ryan, Z.; Salisbury, J.; Kumar, R.

    2006-01-01

    Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered {alpha}-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an {alpha}-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.

  2. The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

    International Nuclear Information System (INIS)

    Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered α-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an α-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin

  3. X-ray crystallography: Assessment and validation of protein-small molecule complexes for drug discovery

    Science.gov (United States)

    Cooper, David R.; Porebski, Przemyslaw J.; Chruszcz, Maksymilian; Minor, Wladek

    2011-01-01

    Introduction Crystallography is the key initial component for structure-based and fragment-based drug design and can often generate leads that can be developed into high potency drugs. Therefore, huge sums of money are committed based on the outcome of crystallography experiments and their interpretation. Areas covered This review discusses how to evaluate the correctness of an X-ray structure, focusing on the validation of small molecule-protein complexes. Various types of inaccuracies found within the PDB are identified and the ramifications of these errors are discussed. The reader will gain an understanding of the key parameters that need to be inspected before a structure can be used in drug discovery efforts, as well as an appreciation of the difficulties of correctly interpreting electron density for small molecules. The reader will also be introduced to methods for validating small molecules within the context of a macromolecular structure. Expert opinion One of the reasons that ligand identification and positioning, within a macromolecular crystal structure, is so difficult is that the quality of small molecules widely varies in the PDB. For this reason, the PDB can not always be considered a reliable repository of structural information pertaining to small molecules, and this makes the derivation of general principles that govern small molecule-protein interactions more difficult. PMID:21779303

  4. Impairment of dendritic cell functions in patients with adaptor protein-3 complex deficiency.

    Science.gov (United States)

    Prandini, Alberto; Salvi, Valentina; Colombo, Francesca; Moratto, Daniele; Lorenzi, Luisa; Vermi, William; De Francesco, Maria Antonia; Notarangelo, Lucia Dora; Porta, Fulvio; Plebani, Alessandro; Facchetti, Fabio; Sozzani, Silvano; Badolato, Raffaele

    2016-06-30

    Hermansky-Pudlak syndrome type 2 (HPS2) is a primary immunodeficiency due to adaptor protein-3 (AP-3) complex deficiency. HPS2 patients present neutropenia, partial albinism, and impaired lysosomal vesicles formation in hematopoietic cells. Given the role of dendritic cells (DCs) in the immune response, we studied monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs) in two HPS2 siblings. Mature HPS2 moDCs showed impaired expression of CD83 and DC-lysosome-associated membrane protein (LAMP), low levels of MIP1-β/CCL4, MIG/CXCL9, and severe defect of interleukin-12 (IL-12) secretion. DCs in lymph-node biopsies from the same patients showed a diffuse cytoplasm reactivity in a large fraction of DC-LAMP(+) cells, instead of the classical dot-like stain. In addition, analysis of pDC-related functions of blood-circulating mononuclear cells revealed reduced interferon-α secretion in response to herpes simplex virus-1 (HSV-1), whereas granzyme-B induction upon IL-3/IL-10 stimulation was normal. Finally, T-cell costimulatory activity, as measured by mixed lymphocyte reaction assay, was lower in patients, suggesting that function and maturation of DCs is abnormal in patients with HPS2.

  5. Determination of amino acid and protein peroxides by the xylenol orange-Fe(III) complex

    International Nuclear Information System (INIS)

    Oxidative stress imposed on living organisms is believed to lead to the depletion of their antioxidant defences, followed by chemical changes in the cell constituents. These may ultimately develop into pathological conditions such as cancer or cardiovascular disease. An assay of peroxides which could be applied to tissues or simple tissue extracts would prove extremely useful in the studies of the phenomenon of oxidative stress. With this purpose, the authors have tested the ability of two peroxide assay techniques to measure the formation of amino acid and protein peroxides in aqueous solutions irradiated with gamma rays, using a modification of the method based on the oxidation of Fe(II)) by peroxides and complexing of the Fe(III) produced by xylenol orange. The molar extinction coefficients of the peroxides tested were determined by comparison with the well-tested iodometric assay. This work was extended to the detection of all organic peroxides in human blood plasma or serum subjected to oxidative stress, where the iodometric assay proved difficult to apply and unreliable because of the binding of iodine to the blood components. Preliminary results suggest that exposure of serum to gamma radiation leads to immediate peroxidation of the proteins, with a delay before generation of lipid peroxides

  6. Protein dynamics revealed in the excitonic spectra of single LH2 complexes

    International Nuclear Information System (INIS)

    The fluorescence emission spectrum of single peripheral light-harvesting (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila exhibits remarkable dynamics on a time scale of several minutes. Often the spectral properties are quasi-stable; sometimes large spectral jumps to the blue or to the red are observed. To explain the dynamics, every pigment is proposed to be in two conformational substates with different excitation energies, which originate from the conformational state of the protein as a result of pigment-protein interaction. Due to the excitonic coupling in the ring of 18 pigments, the two-state assumption generates a substantial amount of distinct spectroscopic states, which reflect part of the inhomogeneous distributed spectral properties of LH2. To describe the observed dynamics, spontaneous and light-induced transitions are introduced between the two states. For each 'realization of the disorder', the spectral properties are calculated using a disordered exciton model combined with the modified Redfield theory to obtain realistic spectral line shapes. The single-molecule fluorescence peak (FLP) distribution, the distribution dependence on the excitation intensity, and the FLP time traces are well described within the framework of this model

  7. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

    DEFF Research Database (Denmark)

    Dou, Q P; Zhao, S; Levin, A H;

    1994-01-01

    By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we...... report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes....... (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide...

  8. The merozoite surface protein 1 complex is a platform for binding to human erythrocytes by Plasmodium falciparum.

    Science.gov (United States)

    Lin, Clara S; Uboldi, Alessandro D; Marapana, Danushka; Czabotar, Peter E; Epp, Christian; Bujard, Hermann; Taylor, Nicole L; Perugini, Matthew A; Hodder, Anthony N; Cowman, Alan F

    2014-09-12

    Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2-4 × 10(-7) m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.

  9. Rybp, a polycomb complex-associated protein, is required for mouse eye development

    Directory of Open Access Journals (Sweden)

    Schreiber-Agus Nicole

    2007-04-01

    Full Text Available Abstract Background Rybp (Ring1 and YY1 binding protein is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of Rybp in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for Rybp in ocular development using four in vivo mouse models which resulted in either the ablation or overexpression of Rybp. Results Our results demonstrate that loss of a single Rybp allele in conventional knockout mice often resulted in retinal coloboma, an incomplete closure of the optic fissure, characterized by perturbed localization of Pax6 but not of Pax2. In addition, about one half of Rybp-/- Rybp+/+ chimeric embryos also developed retinal colobomas and malformed lenses. Tissue-specific transgenic overexpression of Rybp in the lens resulted in abnormal fiber cell differentiation and severe lens opacification with increased levels of AP-2α and Sox2, and reduced levels of βA4-crystallin gene expression. Ubiquitous transgenic overexpression of Rybp in the entire eye caused abnormal retinal folds, corneal neovascularization, and lens opacification. Additional changes included defects in anterior eye development. Conclusion These studies establish Rybp as a novel gene that has been associated with coloboma. Other genes linked to coloboma encode various classes of transcription factors such as BCOR, CBP, Chx10, Pax2, Pax6, Six3, Ski, Vax1 and Vax2. We propose that the multiple functions for Rybp in regulating mouse retinal and lens development are mediated by genetic, epigenetic and physical interactions between these genes and proteins.

  10. Proteins of the origin recognition complex (ORC and DNA topoisomerases on mammalian chromatin

    Directory of Open Access Journals (Sweden)

    Baack Martina

    2009-04-01

    Full Text Available Abstract Background The process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells. Results We have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5 and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin. Conclusion Topoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.

  11. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly

    Science.gov (United States)

    Litovchick, Larisa; Florens, Laurence A.; Swanson, Selene K.; Washburn, Michael P.; DeCaprio, James A.

    2011-01-01

    In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence. PMID:21498570

  12. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly.

    Science.gov (United States)

    Litovchick, Larisa; Florens, Laurence A; Swanson, Selene K; Washburn, Michael P; DeCaprio, James A

    2011-04-15

    In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence. PMID:21498570

  13. Genetic Analysis of Mycobacterium avium Complex Strains Used for Producing Purified Protein Derivatives

    Science.gov (United States)

    Semret, Makeda; Bakker, Douwe; Smart, Nonie; Olsen, Ingrid; Haslov, Kaare; Behr, Marcel A.

    2006-01-01

    For over a century, purified protein derivatives (PPD) have been used to detect mycobacterial infections in humans and livestock. Among these, reagents to detect infections by Mycobacterium avium complex organisms have been produced, but the utility of these reagents has not been clearly established due in part to limited biologic and immunologic standardization. Because there is little information about the strains used to produce these reagents (avian PPD, intracellulare PPD, scrofulaceum PPD, and Johnin), we have performed genetic characterizations of strains used to produce these products. Sequence analysis of 16S rRNA and the hsp65 gene provided results concordant with species designations provided for M. avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum organisms. For M. avium strains, comparative genomic hybridization was performed on a whole-genome DNA microarray, revealing one novel 7.9-kilobase genomic deletion in certain Johnin-producing strains, in addition to genomic variability inherent to the particular M. avium subspecies. Our findings indicate that considerable genomic differences exist between organisms used for reagents and the infecting organism being studied. These results serve as a baseline for potency studies of different preparations and should aid in comparative studies of newly discovered antigens for the diagnosis of infection and disease by M. avium complex organisms. PMID:16960109

  14. Ena/VASP proteins cooperate with the WAVE complex to regulate the actin cytoskeleton.

    Science.gov (United States)

    Chen, Xing Judy; Squarr, Anna Julia; Stephan, Raiko; Chen, Baoyu; Higgins, Theresa E; Barry, David J; Martin, Morag C; Rosen, Michael K; Bogdan, Sven; Way, Michael

    2014-09-01

    Ena/VASP proteins and the WAVE regulatory complex (WRC) regulate cell motility by virtue of their ability to independently promote actin polymerization. We demonstrate that Ena/VASP and the WRC control actin polymerization in a cooperative manner through the interaction of the Ena/VASP EVH1 domain with an extended proline rich motif in Abi. This interaction increases cell migration and enables VASP to cooperatively enhance WRC stimulation of Arp2/3 complex-mediated actin assembly in vitro in the presence of Rac. Loss of this interaction in Drosophila macrophages results in defects in lamellipodia formation, cell spreading, and redistribution of Ena to the tips of filopodia-like extensions. Rescue experiments of abi mutants also reveals a physiological requirement for the Abi:Ena interaction in photoreceptor axon targeting and oogenesis. Our data demonstrate that the activities of Ena/VASP and the WRC are intimately linked to ensure optimal control of actin polymerization during cell migration and development.

  15. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    Directory of Open Access Journals (Sweden)

    Benjamin Clémençon

    2012-02-01

    Full Text Available The existence of a mitochondrial interactosome (MI has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp and inorganic phosphate (PiC carriers as well as the VDAC (or mitochondrial porin catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1 under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003.

  16. Heat shock protein 90 (Hsp90) chaperone complex. A molecular target for enhancement of thermosensitivity and radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Akimoto, Tetsuo; Nonaka, Tetsuo; Kitamoto, Yoshizumi; Sakurai, Hideyuki [Gunma Univ., Maebashi (Japan). School of Medicine; Mitsuhashi, Norio [Tokyo Women' s Medical Coll. (Japan)

    2002-09-01

    Heat shock protein 90 (Hsp90) is a highly conserved heat shock protein in animal and plants, and exists abundantly in the cytoplasm in unstressed condition, accounting for 1-2% in cytoplasmic proteins. Main difference of Hsp90 from other Hsps are its substrate that Hsp90 binds to. These substrates include various signal transduction proteins, kinase, steroid receptors and transcription factors, therefore, Hsp90 plays a key role in maintaining cellular signal transduction networks. Many chaperoned proteins (client proteins) of Hsp90 are associated with cellular proliferation or malignant transformation, thus Hsp90 chaperone complex has been focused as targets for cancer therapy. Among the client proteins, there are several molecules that have been defined as targets or factors for determination or enhancement of radiosensitivity or thermosensitivity. Thus, it is easily speculated that Hsp90 chaperone complex inhibitors that disrupt association of Hsp90 and client protein in combination with radiation or/and heat has potential effect on enhancement of radiosensitivity or thermosensitivity. In this paper, possible mechanisms in enhancing radiosensitivity or thermosensitivity according to the client proteins will be summarized. (author)

  17. Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17.

    Science.gov (United States)

    Howard, Paul W; Jue, Shall F; Maurer, Richard A

    2013-08-15

    Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known components of the IFT B complex. In addition, peptides corresponding to mouse tetratricopeptide repeat proteins, TTC30A1, TTC30A2 and TTC30B were identified. The mouse Ttc30A1, Ttc30A2, Ttc30B genes are orthologs of Caenorhabditis elegans dyf-1, which is required for assembly of the distal segment of the cilia. We used co-immunoprecipitation studies to provide evidence that, TTC30A1, TTC30A2 or TTC30B can be incorporated into a complex with a known IFT B protein, IFT52. We also found that TTC30B can interact with mouse KIF17, a kinesin which participates in IFT. In vitro expression in a cell-free system followed by co-immunoprecipitation also provided evidence that TTC30B can directly interact with several different IFT B complex proteins. The findings support the view that mouse TTC30A1, TTC30A2 and TTC30B can contribute to the IFT B complex, likely through interactions with multiple IFT proteins and also suggest a possible link to the molecular motor, KIF17 to support transport of cargo during IFT. PMID:23810713

  18. Nucleolar protein PinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA

    OpenAIRE

    Lin, Jue; Elizabeth H. Blackburn

    2004-01-01

    Human TRF1-binding protein PinX1 inhibits telomerase activity. Here we report that overexpression of yeast PinX1p (yPinX1p) results in shortened telomeres and decreased in vitro telomerase activity. yPinX1p coimmunoprecipitated withyeast telomerase protein Est2p even in cells lacking the telomerase RNA TLC1, or the telomerase-associated proteins Est1p and Est3p. Est2p regions required for binding to yPinX1p or TLC1 were similar. Furthermore, we found two distinct Est2p complexes exist, contai...

  19. Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes.

    Science.gov (United States)

    Brabec, Viktor; Kasparkova, Jana; Kostrhunova, Hana; Farrell, Nicholas P

    2016-01-01

    Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes. PMID:27574114

  20. Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes.

    Science.gov (United States)

    Lössl, Philip; Sinz, Andrea

    2016-01-01

    During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this "traditional" cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.

  1. Exploiting gene deletion fitness effects in yeast to understand the modular architecture of protein complexes under different growth conditions

    Directory of Open Access Journals (Sweden)

    Babu M Madan

    2009-07-01

    Full Text Available Abstract Background Understanding how individual genes contribute towards the fitness of an organism is a fundamental problem in biology. Although recent genome-wide screens have generated abundant data on quantitative fitness for single gene knockouts, very few studies have systematically integrated other types of biological information to understand how and why deletion of specific genes give rise to a particular fitness effect. In this study, we combine quantitative fitness data for single gene knock-outs in yeast with large-scale interaction discovery experiments to understand the effect of gene deletion on the modular architecture of protein complexes, under different growth conditions. Results Our analysis reveals that genes in complexes show more severe fitness effects upon deletion than other genes but, in contrast to what has been observed in binary protein-protein interaction networks, we find that this is not related to the number of complexes in which they are present. We also find that, in general, the core and attachment components of protein complexes are equally important for the complex machinery to function. However, when quantifying the importance of core and attachments in single complex variations, or isoforms, we observe that this global trend originates from either the core or the attachment components being more important for strain fitness, both being equally important or both being dispensable. Finally, our study reveals that different isoforms of a complex can exhibit distinct fitness patterns across growth conditions. Conclusion This study presents a powerful approach to unveil the molecular basis for various complex phenotypic profiles observed in gene deletion experiments. It also highlights some interesting cases of potential functional compensation between protein paralogues and suggests a new piece to fit into the histone-code puzzle.

  2. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    Science.gov (United States)

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root.

  3. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    Science.gov (United States)

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root. PMID:27457987

  4. Computational study of ligand binding in lipid transfer proteins: Structures, interfaces, and free energies of protein-lipid complexes

    OpenAIRE

    Fernandez Pacios, Luis; Gomez Casado, Cristina; Tordesillas Villuendas, Leticia; Palacín Gómez, Aranzazu; Sanchez-Monge Laguna De Rins, Maria Rosa; Díaz Perales, Araceli

    2012-01-01

    Plant nonspecific lipid transfer proteins (nsLTPs) bind a wide variety of lipids, which allows them to perform disparate functions. Recent reports on their multifunctionality in plant growth processes have posed new questions on the versatile binding abilities of these proteins. The lack of binding specificity has been customarily explained in qualitative terms on the basis of a supposed structural flexibility and nonspecificity of hydrophobic protein-ligand interactions. We present here a co...

  5. Isolation of complexes formed between insulin-like growth factor-binding protein-3 and transferrin from the human serum

    Directory of Open Access Journals (Sweden)

    Miljuš Goran

    2012-01-01

    Full Text Available Insulin-like growth factors (IGFs play an important role in the regulation of cell growth, differentiation and metabolism. The amount of free, biologically active IGFs is regulated by the IGF-binding proteins (IGFBPs. IGFBP-3 is the most abundant binding protein and it is known to interact with other circulating proteins, including transferrin (Tf. In order to elucidate the possible role of IGF/IGFBP-3 in the iron metabolism, it is necessary to isolate IGFBP-3/Tf complexes. Several affinity-based techniques were employed. Results have shown that only double immunoprecipitation method with anti-Tf and anti-IGFBP-3 antibodies selectively separated complexes from other molecular forms, such as monomers, oligomers or fragments of IGFBP-3 and Tf. Isolated complexes can now be used to investigate the relationship between IGF/IGFBP-3 and iron, both in structural and metabolic tеrms.

  6. Linear motif-mediated interactions have contributed to the evolution of modularity in complex protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Inhae Kim

    2014-10-01

    Full Text Available The modular architecture of protein-protein interaction (PPI networks is evident in diverse species with a wide range of complexity. However, the molecular components that lead to the evolution of modularity in PPI networks have not been clearly identified. Here, we show that weak domain-linear motif interactions (DLIs are more likely to connect different biological modules than strong domain-domain interactions (DDIs. This molecular division of labor is essential for the evolution of modularity in the complex PPI networks of diverse eukaryotic species. In particular, DLIs may compensate for the reduction in module boundaries that originate from increased connections between different modules in complex PPI networks. In addition, we show that the identification of biological modules can be greatly improved by including molecular characteristics of protein interactions. Our findings suggest that transient interactions have played a unique role in shaping the architecture and modularity of biological networks over the course of evolution.

  7. Low Charge and Reduced Mobility of Membrane Protein Complexes Has Implications for Calibration of Collision Cross Section Measurements.

    Science.gov (United States)

    Allison, Timothy M; Landreh, Michael; Benesch, Justin L P; Robinson, Carol V

    2016-06-01

    Ion mobility mass spectrometry of integral membrane proteins provides valuable insights into their architecture and stability. Here we show that, due to their lower charge, the average mobility of native-like membrane protein ions is approximately 30% lower than that of soluble proteins of similar mass. This has implications for drift time measurements, made on traveling wave ion mobility mass spectrometers, which have to be calibrated to extract collision cross sections (Ω). Common calibration strategies employ unfolded or native-like soluble protein standards with masses and mobilities comparable to the protein of interest. We compare Ω values for membrane proteins, derived from standard calibration protocols using soluble proteins, to values measured using an RF-confined drift tube. Our results demonstrate that, while common calibration methods underestimate Ω for native-like or unfolded membrane protein complexes, higher mass soluble calibration standards consistently yield more accurate Ω values. These findings enable us to obtain directly structural information for highly charge-reduced complexes by traveling wave ion mobility mass spectrometry. PMID:27153188

  8. Structure of a membrane-attack complex/perforin (MACPF) family protein from the human gut symbiont Bacteroides thetaiotaomicron

    International Nuclear Information System (INIS)

    The crystal structure of a novel MACPF protein, which may play a role in the adaptation of commensal bacteria to host environments in the human gut, was determined and analyzed. Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT-3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment

  9. Toll-like receptor signaling in vertebrates: testing the integration of protein, complex, and pathway data in the protein ontology framework.

    Directory of Open Access Journals (Sweden)

    Cecilia Arighi

    Full Text Available The Protein Ontology (PRO provides terms for and supports annotation of species-specific protein complexes in an ontology framework that relates them both to their components and to species-independent families of complexes. Comprehensive curation of experimentally known forms and annotations thereof is expected to expose discrepancies, differences, and gaps in our knowledge. We have annotated the early events of innate immune signaling mediated by Toll-Like Receptor 3 and 4 complexes in human, mouse, and chicken. The resulting ontology and annotation data set has allowed us to identify species-specific gaps in experimental data and possible functional differences between species, and to employ inferred structural and functional relationships to suggest plausible resolutions of these discrepancies and gaps.

  10. A Phospholipid-Protein Complex from Krill with Antioxidative and Immunomodulating Properties Reduced Plasma Triacylglycerol and Hepatic Lipogenesis in Rats

    OpenAIRE

    Marie S. Ramsvik; Bodil Bjørndal; Inge Bruheim; Pavol Bohov; Berge, Rolf K.

    2015-01-01

    Dietary intake of marine omega-3 polyunsaturated fatty acids (n-3 PUFAs) can change the plasma profile from atherogenic to cardioprotective. In addition, there is growing evidence that proteins of marine origin may have health benefits. We investigated a phospholipid-protein complex (PPC) from krill that is hypothesized to influence lipid metabolism, inflammation, and redox status. Male Wistar rats were fed a control diet (2% soy oil, 8% lard, 20% casein), or diets where corresponding amount...

  11. Simian and Human Immunodeficiency Virus Nef Proteins Use Different Surfaces To Downregulate Class I Major Histocompatibility Complex Antigen Expression

    OpenAIRE

    Swigut, Tomek; Iafrate, A. John; Muench, Jan; Kirchhoff, Frank; Skowronski, Jacek

    2000-01-01

    Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregula...

  12. Comparison of displacement versus gradient mode for separation of a complex protein mixture by anion-exchange chromatography.

    Science.gov (United States)

    Ahrends, Robert; Lichtner, Björn; Buck, Friedrich; Hildebrand, Diana; Kotasinska, Marta; Kohlbacher, Oliver; Kwiatkowski, Marcel; Wagner, Moritz; Trusch, Maria; Schlüter, Hartmut

    2012-07-15

    Liquid chromatography is often the method of choice for the analysis of proteins in their native state. Nevertheless compared to two-dimensional electrophoresis, the resolution of common chromatographic techniques is low. Liquid chromatography in the displacement mode has previously been shown to offer higher resolution and to elute proteins in the high concentrations. In this study we compared to what extend displacement mode was a suitable alternative to gradient mode for the separation of a complex protein mixture using anion-exchange displacement chromatography and if it is therefore helpful for proteomic investigations. Hence we analyzed the qualitative protein composition of each fraction by tryptic digestion of the proteins, analysis of the tryptic peptides by liquid chromatography coupled to mass spectrometry followed by data base analysis and by measuring the elution profiles of 22 selected proteins with selected reaction monitoring mass spectrometry. In the fractions of displacement mode a significantly higher number of identified proteins (51 versus 16) was yielded in comparison to gradient mode. The resolution of displacement chromatography was slightly lower than of gradient chromatography for many but not for all proteins. The selectivities of displacement mode and gradient mode are very different. In conclusion displacement chromatography is a well suited alternative for top-down proteomic approaches which start with separating intact proteins first prior to mass spectrometric analysis of intact or digested proteins. The significant orthogonality of both modes may be used in the future for combining them in multidimensional fractionation procedures. PMID:22727752

  13. Seasonal oscillation of liver-derived hibernation protein complex in the central nervous system of non-hibernating mammals

    OpenAIRE

    Seldin, Marcus M.; Byerly, Mardi S.; Petersen, Pia S.; Swanson, Roy; Balkema-Buschmann, Anne; Martin H. Groschup; Wong, G. William

    2014-01-01

    Mammalian hibernation elicits profound changes in whole-body physiology. The liver-derived hibernation protein (HP) complex, consisting of HP-20, HP-25 and HP-27, was shown to oscillate circannually, and this oscillation in the central nervous system (CNS) was suggested to play a role in hibernation. The HP complex has been found in hibernating chipmunks but not in related non-hibernating tree squirrels, leading to the suggestion that hibernation-specific genes may underlie the origin of hibe...

  14. Basic domains target protein subunits of the RNase MRP complex to the nucleolus independently of complex association.

    NARCIS (Netherlands)

    Eenennaam, H. van; Heijden, A.G. van der; Janssen, R.J.T.; Venrooij, W.J.W. van; Pruijn, G.J.M.

    2001-01-01

    The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both R

  15. Chemical shifts assignments of the archaeal MC1 protein and a strongly bent 15 base pairs DNA duplex in complex.

    Science.gov (United States)

    Loth, Karine; Landon, Céline; Paquet, Françoise

    2015-04-01

    MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55 in laboratory growth conditions and is structurally unrelated to other DNA-binding proteins. MC1 functions are to shape and to protect DNA against thermal denaturation by binding to it. Therefore, MC1 has a strong affinity for any double-stranded DNA. However, it recognizes and preferentially binds to bent DNA, such as four-way junctions and negatively supercoiled DNA minicircles. Combining NMR data, electron microscopy data, biochemistry, molecular modelisation and docking approaches, we proposed recently a new type of DNA/protein complex, in which the monomeric protein MC1 binds on the concave side of a strongly bent 15 base pairs DNA. We present here the NMR chemical shifts assignments of each partner in the complex, (1)H (15)N MC1 protein and (1)H (13)C (15)N bent duplex DNA, as first step towards the first experimental 3D structure of this new type of DNA/protein complex.

  16. Mitochondrial complex I protein differs among residual feed intake phenotype in beef cattle.

    Science.gov (United States)

    Ramos, M H; Kerley, M S

    2013-07-01

    Four experiments were performed to determine if residual feed intake (RFI) was related to mitochondrial complex I (CI) protein. For Exp. 1, crossbred Angus steers (initial BW 270 ± 2.0 kg) were fed for a total of 170 d (n = 72). For Exp. 2, crossbred Braunvieh steers (initial BW 280 ± 3.0 kg) were fed for a total of 150 d (n = 50). For Exp. 3, crossbred Braunvieh heifers (initial BW 260 ± 3.0 kg) were fed for a total of 160 d (n = 40). For Exp. 4, crossbred Angus steers (initial BW 290 ± 3.0 kg) were fed for a total of 160 d (n = 40). All cattle in all experiments were fed the same diet. The variable RFI was calculated as the difference between predicted and actual DMI. Predicted DMI was calculated from regressing intake on ADG and metabolic body weight. Blood was collected, lymphocytes were isolated, and antibody used to capture CI. For Exp. 1, 2, and 3, CI quantity was measured using an ELISA commercial kit (Mitosciences, OR). For Exp. 4, CI subunits were separated by gel electrophoresis and bands were analyzed for differences in concentration (absorbance) among RFI phenotypes. For all 4 experiments, there was a significant difference (P 0.05) was reported for ADG and metabolic midweight. For Exp. 1, 2, and 3, CI concentration in mitochondria was greater (P < 0.05) for low RFI compared with other treatments. For Exp. 4, animals with low RFI had a trend (P = 0.07) for greater concentration of Band I (protein S1) than high RFI. Correlation between RFI and Band I was -0.72 (P = 0.04). We concluded that mitochondrial function was at least in part responsible for differences among animals in metabolic efficiency.

  17. Theoretical Simulations and Ultrafast Pump-probe Spectroscopy Experiments in Pigment-protein Photosynthetic Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Buck, D.R.

    2000-09-12

    Theoretical simulations and ultrafast pump-probe laser spectroscopy experiments were used to study photosynthetic pigment-protein complexes and antennae found in green sulfur bacteria such as Prosthecochloris aestuarii, Chloroflexus aurantiacus, and Chlorobium tepidum. The work focused on understanding structure-function relationships in energy transfer processes in these complexes through experiments and trying to model that data as we tested our theoretical assumptions with calculations. Theoretical exciton calculations on tubular pigment aggregates yield electronic absorption spectra that are superimpositions of linear J-aggregate spectra. The electronic spectroscopy of BChl c/d/e antennae in light harvesting chlorosomes from Chloroflexus aurantiacus differs considerably from J-aggregate spectra. Strong symmetry breaking is needed if we hope to simulate the absorption spectra of the BChl c antenna. The theory for simulating absorption difference spectra in strongly coupled photosynthetic antenna is described, first for a relatively simple heterodimer, then for the general N-pigment system. The theory is applied to the Fenna-Matthews-Olson (FMO) BChl a protein trimers from Prosthecochloris aestuarii and then compared with experimental low-temperature absorption difference spectra of FMO trimers from Chlorobium tepidum. Circular dichroism spectra of the FMO trimer are unusually sensitive to diagonal energy disorder. Substantial differences occur between CD spectra in exciton simulations performed with and without realistic inhomogeneous distribution functions for the input pigment diagonal energies. Anisotropic absorption difference spectroscopy measurements are less consistent with 21-pigment trimer simulations than 7-pigment monomer simulations which assume that the laser-prepared states are localized within a subunit of the trimer. Experimental anisotropies from real samples likely arise from statistical averaging over states with diagonal energies shifted by

  18. Two-dimensional blue native/SDS-PAGE analysis of whole cell lysate protein complexes of rice in response to salt stress.

    Science.gov (United States)

    Hashemi, Amenehsadat; Gharechahi, Javad; Nematzadeh, Ghorbanali; Shekari, Faezeh; Hosseini, Seyed Abdollah; Salekdeh, Ghasem Hosseini

    2016-08-01

    To understand the biology of a plant in response to stress, insight into protein-protein interactions, which almost define cell behavior, is thought to be crucial. Here, we provide a comparative complexomics analysis of leaf whole cell lysate of two rice genotypes with contrasting responses to salt using two-dimensional blue native/SDS-PAGE (2D-BN/SDS-PAGE). We aimed to identify changes in subunit composition and stoichiometry of protein complexes elicited by salt. Using mild detergent for protein complex solubilization, we were able to identify 9 protein assemblies as hetero-oligomeric and 30 as homo-oligomeric complexes. A total of 20 proteins were identified as monomers in the 2D-BN/SDS-PAGE gels. In addition to identifying known protein complexes that confirm the technical validity of our analysis, we were also able to discover novel protein-protein interactions. Interestingly, an interaction was detected for glycolytic enzymes enolase (ENO1) and triosephosphate isomerase (TPI) and also for a chlorophyll a-b binding protein and RuBisCo small subunit. To show changes in subunit composition and stoichiometry of protein assemblies during salt stress, the differential abundance of interacting proteins was compared between salt-treated and control plants. A detailed exploration of some of the protein complexes provided novel insight into the function, composition, stoichiometry and dynamics of known and previously uncharacterized protein complexes in response to salt stress. PMID:27362847

  19. Inducer effect on the complex formation between rat liver nuclear proteins and cytochrome P450 2B gene regulatory elements.

    Science.gov (United States)

    Duzhak, T G; Schwartz, E I; Gulyaeva, L F; Lyakhovich, V V

    2002-09-01

    DNA gel retardation assay has been applied to the investigation of complexes between rat liver nuclear proteins and Barbie box positive regulatory element of cytochrome P450 2B (CYP2B) genes. The intensities of B1 and B2 bands detected in the absence of an inducer increased after 30 min protein incubation with phenobarbital (PB) or triphenyldioxane (TPD), but not with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOPOB). In addition, a new complex (B3 band) was for the first time detected under induction by PB, TPD, and TCPOPOB. Increase in the incubation time up to 2 h facilitated the formation of other new complexes (B4 and B5 bands), which were detected only in the presence of TPD. The use of [3H]TPD in hybridization experiments revealed that this inducer, capable of binding to Barbie box DNA, is also present in B4 and B5 complexes. It is probable that the investigated compounds activate the same proteins at the initial induction steps, which correlates with the formation of B1, B2, and B3 complexes. The further induction step might be inducer-specific, as indicated by the formation of B4 and B5 complexes in the presence of TPD only. Thus, the present data suggest the possibility of specific gene activation signaling pathways that are dependent on a particular inducer. PMID:12387719

  20. Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

    Directory of Open Access Journals (Sweden)

    Jaqueline Sudiman

    Full Text Available Developmental competence of in vitro matured (IVM oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15 or growth differentiation factor (GDF9 to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(PH, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2% compared to controls (43.3±2.4%, 28.9±3.7% and to mature GDF9+FSH (36.1±3.0%. The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(PH, and reduced glutathione (GSH levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.